Anti-ror2 antibodies, antibody fragments, their immunoconjucates and uses thereof

ABSTRACT

A polypeptide having a heavy chain variable region and/or light chain variable region that specifically binds to Ror2 protein as well as antibodies and antibody fragments containing the heavy chain variable region and/or the light chain variable region that bind to Ror2 protein. Pharmaceutical compositions and kits comprising the polypeptide or antibodies and antibody fragments containing the polypeptide are also provided

FIELD OF THE DISCLOSURE

This disclosure relates anti-Ror2 antibodies, antibody fragments andimmunoconjugates of such antibodies and antibody fragments and uses ofthe antibodies, antibody fragments and immunoconjugates in diagnosticand therapeutic methods.

BACKGROUND OF THE DISCLOSURE

Receptor tyrosine kinases (RTKs) are a family of cell surface receptorsthat regulate a range of normal cellular processes throughligand-controlled tyrosine kinase activity. Over the past 20 years,deregulation of RTKs has been shown to play a critical role in cancerdevelopment and progression. RTKs are now recognized as prognosticmolecular biomarkers and as targets of oncology therapeutics.

Ror2, also called receptor tyrosine kinase-like orphan receptor 2, is amembrane-bound RTK that is activated by non-canonical Wnt signalingthrough its association with the Wnt5A glycoprotein during normal boneand cartilage development. Ror2 has only one transmembrane domain, whichseparates its extracellular and intracellular domains (FIG. 1). Ror2 isknown to play crucial roles in the normal development of various organsand tissues. In mammals, Ror2- and Wnt5A-deficient mice exhibit similarabnormalities during developmental morphogenesis, reflecting theirdefects in convergent extension movements and planar cell polarity.Furthermore, mutations of the human Ror2 gene are responsible for thegenetic skeletal disorders dominant brachydactyly type B and recessiveRobinow syndrome. Ror2 has been found to mediate polarized cellmigration and malfunction of Ror2 results in heritable skeletaldisorders and tumor invasion (Minami et al., “Ror-family receptortyrosine kinases in noncanonical Wnt signaling: their implications indevelopmental morphogenesis and human diseases,” Dev Dyn., vol. 239, pp.1-15, 2010).

Ror2 has also been reported to have pro-tumorigenic effects. US2014/0322234 discloses that the expression and activity of Ror2 invarious cancers is different from normal tissues. Thus, it is suggestedthat dysregulation of Ror2 plays a role in the pathogenesis of a varietyof human cancers. US 2014/0322234 also contemplates that antibodiesagainst Ror2 may be used in diagnosis of cancers and inhibition ofcancer cell growth. For example, such antibodies may be conjugated to acytotoxic agent that has a high degree of cytotoxicity for cancer cellsexpressing Ror2, such that the cytotoxic agent can effectively kill thecancer cells. The Ror2 gene may also be used in classification ofcancers according to the Ror2 expression pattern in the cancers.

Ford et al. (“The dual role of the novel Wnt receptor tyrosine kinase,Ror2, in human carcinogenesis,” International Journal of Cancer, vol.133, pp. 779-787, 2013) further explores the mechanism of Ror2 incarcinogenesis. This reference discloses that Ror2 is involved in thedevelopment and progression of cancers. Specifically, Ror2 has beenfound to play a pivotal role in carcinogenesis of numerous cancersincluding colon cancer, hepatocellular carcinoma, metastatic melanomaand renal cell carcinoma. For example, Ror2 is over-expressed inosteosarcoma, melanoma, renal cell carcinoma, prostate carcinoma,squamous cell carcinomas of the head and neck and stromal tumors. Ror2thus has the potential of being a drug target for cancer treatments byinhibition of the Wnt signaling pathway.

Further, Debebe et al., (“Ror2 as a therapeutic target in cancer,”Pharmacol. Ther., vol. 50, pp. 143-148, 2015) discloses that Ror2mediates both canonical and non-canonical signaling pathways. Ror2 ishighly expressed in osteosarcoma and renal cell carcinomas, as well asin melanoma, colon cancer, squamous cell carcinoma of the head and neck,and breast cancer. In the majority of these cancer types, Ror2expression is associated with more aggressive cancer states. Thus, thisreference also suggests that Ror2 is a potential target for cancertreatment.

Though monoclonal antibodies against Ror2 are commercially available,anti-Ror2 antibodies suitable for cancer therapy have not been reported.The present invention provides anti-Ror2 antibodies or antibodyfragments that are suitable for therapeutic and diagnostic use,especially for diagnosis and treatment of cancers. Some of the anti-Ror2antibodies or antibody fragments have a higher binding affinity to Ror2in a tumor in comparison with Ror2 present in normal tissue. Theseanti-Ror2 antibodies or antibody fragments of the present invention haveat least comparable efficacy as well as a longer half-life, but reducedside-effects, in comparison with monoclonal anti-Ror2 antibodies knownin the art. This may permit use of higher dosages of these anti-Ror2antibodies or antibody fragments, thus providing a more effectivetherapeutic option without a corresponding significant increase in sideeffects.

SUMMARY OF THE DISCLOSURE

In one aspect, the present invention provides an isolated heavy chainvariable region polypeptide that specifically binds to the Ror2 protein.The polypeptide includes three complementarity determining regions H1,H2, and H3 sequences, wherein:

-   -   the H1 sequence is GYTX₁TEX₂X₃X₄H (SEQ ID NO:1) or GYSITTGX₂₉YWN        (SEQ ID NO:4);    -   the H2 sequence is X₅X₆X₇X₈NNGGTGYNQKFKG (SEQ ID NO:2) or        YITYDGSX₃₀NYNPSLKN (SEQ ID NO:5); and    -   the H3 sequence is X₉X₁₀X₁₁SX₁₂YX₁₃YX₁₄X₁₅SYFX₁₆X₁₇X₁₈ (SEQ ID        NO:3) or CSX₃₁X₃₂X₃₃X₃₄VX₃₅X₃₆X₃₇LDX₃₈ (SEQ ID NO:6);

wherein

X₁ is F or E,

X₂ is Y or D,

X₃ is T or C,

X₄ is M or D or E or Y,

X₅ is G or S,

X₆ is I or E,

X₇ is N or C or L or V,

X₈ is T or D or E,

X₉ is A or M or T,

X₁₀ is R or H,

X₁₁ is G or E,

X₁₂ is L or F,

X₁₃ is S or G,

X₁₄ is G or D,

X₁₅ is N or E,

X₁₆ is D or L,

X₁₇ is Y or C or T,

X₁₈ is W or L,

X₂₉ is Y or E or R or T,

X₃₀ is K or N,

X₃₁ is R or G or H or W or Y,

X₃₂ is F or C or N or Q,

X₃₃ is E or S,

X₃₄ is G or E or F or H or M or Q or S,

X₃₅ is W or A or I or P or Q or T or V,

X₃₆ is Y or G or N or Q,

X₃₇ is G or S or T, and

X₃₈ Y or I.

In another aspect, this isolated heavy chain variable region polypeptideis combined with an isolated light chain variable region that includesthree complementarity determining regions L1, L2, and L3 sequences,wherein:

-   -   the L1 sequence is SATSSX₁₉X₂₉X₂₁MX₂₂ (SEQ ID NO:7) or        RASESVDRYGNSX₃₉IH (SEQ ID NO:10);    -   the L2 sequence is X₂₃TSNLAS (SEQ ID NO:8) or X₄₀TYX₄₁LES (SEQ        ID NO:11); and    -   the L3 sequence is QX₂₄X₂₅SX₂₆YPFX₂₇X₂₈ (SEQ ID NO:9) or        QQX₄₂NX₄₃DPX₄₄TX₄₅ (SEQ ID NO:12);

wherein

X₁₉ is V or E,

X₂₀ is S or D,

X₂₁ is Y or C or D,

X₂₂ is H or G or L,

X₂₃ is G or C or H or P,

X₂₄ is Q or E,

X₂₅ is R or H,

X₂₆ is S or D or G or I or Q or V,

X₂₇ is T or D,

X₂₈ is F or D or E,

X₃₉ is F or S or T,

X₄₀ is R or C or D or E or W,

X₄₁ is N or D,

X₄₂ is T or I or P,

X₄₃ is E or V,

X₄₄ is W or T, and

X₄₅ is F or T.

In yet another aspect, the present invention provides an anti-Ror2antibody or antibody fragment that includes the isolated heavy chainvariable region polypeptide of the invention.

In yet another aspect, the present invention provides an immunoconjugatethat includes the antibody or antibody fragment of the invention,optionally conjugated to an agent selected from a chemotherapeuticagent, a radioactive atom, a cytostatic agent and a cytotoxic agent.

In yet another aspect, the present invention provides a pharmaceuticalcomposition that includes the polypeptide, the antibody or antibodyfragment, or the immunoconjugate of the invention, together with apharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a kit fordiagnosis or treatment including the polypeptide, the antibody orantibody fragment, or the immunoconjugate of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the structure of human Ror2 protein.The protein contains an Ig-like domain (Ig), a frizzled or cysteine-rich(CRD) domain, and a kringle (Kr) domain in the extracellular domain. Theextracellular and intracellular domains are separated by a transmembrane(TM) domain. The intracellular domain contains a tyrosine kinase (TK)domain and a proline-rich domain (PR) flanked by serine/threonine (ST)rich domains.

FIGS. 2A-2B show sequence alignments of exemplary heavy chain variableregions of anti-Ror2 antibodies of the present invention.

FIGS. 3A-3B show sequence alignments of exemplary light chain variableregions of anti-Ror2 antibodies of the present invention.

FIG. 4 shows a size exclusion chromatograph indicating that theanti-Ror2 antibodies of the invention do not aggregate, as described inExample 1.

FIG. 5 shows pH-dependent binding profiles of anti-Ror2 antibodies ofthe present invention for binding to Ror2, as described in Example 1.

FIGS. 6A-6B show on and off rates of conditionally active antibodies ofthe invention as measured by surface plasmon resonance (SPR) assay, asdescribed in Example 1.

FIGS. 7A-7B show effects on tumor volume of treatment of xenograftedmice with a paclitaxel-conjugated anti-Ror2 antibody of the presentinvention, as described in Example 2.

FIG. 8 shows the pH dependent binding affinity of an exemplaryconditionally active antibody BAP048 measured by pH titration.

FIG. 9 shows the binding affinity of the conditionally active antibodyBAP048 to Ror2 proteins of human, cynomolgus, and mouse.

FIG. 10 shows cell killing of the conditionally active antibody BAP048conjugated to Monomethyl auristatin E (MMAE) on HEK293 cells expressinghuman Ror2.

FIGS. 11A-11C show cell killing of the conditionally active antibodyBAP048 conjugated to MMAE on LCLC103H cells.

FIGS. 12A-12C show cell killing of the conditionally active antibodyBAP048 conjugated to MMAE on HT1080 cells.

FIG. 13 shows treatment of mouse tumors induced by LCLC103H using theconditionally active antibody BAP048 conjugated to MMAE.

FIG. 14 shows treatment of mouse tumors induced by LCLC103H using theconditionally active antibody BAP048 conjugated to MMAE throughdifferent linkers.

FIGS. 15A-15B show treatment of mouse tumors induced by HT1080 orMDA-MB-436 respectively, using the conditionally active antibody BAP048conjugated to MMAE.

DEFINITIONS

In order to facilitate understanding of the examples provided herein,certain frequently occurring terms are defined herein.

In connection with a measured quantity, the term “about” as used hereinrefers to the normal variation in that measured quantity that would beexpected by a skilled person making the measurement and exercising alevel of care commensurate with the objective of the measurement and theprecision of the measuring equipment used. Unless otherwise indicated,“about” refers to a variation of +/−10% of the value provided.

The term “affinity” as used herein refers to the strength of the sumtotal of noncovalent interactions between a single binding site of amolecule (e.g., an antibody) and its binding partner (e.g., an antigen).Unless indicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Specificillustrative and exemplary embodiments for measuring binding affinityare described in the following.

The term “affinity matured” antibody as used herein refers to anantibody with one or more alterations in one or more hypervariableregions (HVRs), compared to a parent antibody which does not possesssuch alterations, such alterations resulting in an improvement in theaffinity of the antibody for antigen.

The term “amino acid” as used herein refers to any organic compound thatcontains an amino group (—NH2) and a carboxyl group (—COOH); preferablyeither as free groups or alternatively after condensation as part ofpeptide bonds. The “twenty naturally encoded polypeptide-formingalpha-amino acids” are understood in the art and refer to: alanine (alaor A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp orD), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin orQ), glycine (gly or G), histidine (his or H), isoleucine (ile or I),leucine (leu or L), lysine (lys or K), methionine (met or M),phenylalanine (phe or F), proline (pro or P), serine (ser or S),threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), andvaline (val or V).

The term “antibody” as used herein refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)2, Fv, and SCA fragments, that are capable of bindingto an epitope of an antigen. These antibody fragments, which retain someability to selectively bind to an antigen (e.g., a polypeptide antigen)of the antibody from which they are derived, can be made using wellknown methods in the art (see, e.g., Harlow and Lane, supra), and aredescribed further, as follows. Antibodies can be used to isolatepreparative quantities of the antigen by immunoaffinity chromatography.Various other uses of such antibodies are to diagnose and/or stagedisease (e.g., neoplasia) and for therapeutic application to treatdisease, such as for example: neoplasia, autoimmune disease, AIDS,cardiovascular disease, infections, and the like. Chimeric, human-like,humanized or fully human antibodies are particularly useful foradministration to human patients.

An Fab fragment consists of a monovalent antigen-binding fragment of anantibody molecule, and can be produced by digestion of a whole antibodymolecule with the enzyme papain, to yield a fragment consisting of anintact light chain and a portion of a heavy chain.

An Fab′ fragment of an antibody molecule can be obtained by treating awhole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

An (Fab′)2 fragment of an antibody can be obtained by treating a wholeantibody molecule with the enzyme pepsin, without subsequent reduction.A (Fab′)2 fragment is a dimer of two Fab′ fragments, held together bytwo disulfide bonds.

An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

The term “antibody fragment” as used herein refers to a molecule otherthan an intact antibody that comprises a portion of an intact antibodythat binds the antigen to which the intact antibody binds. Examples ofantibody fragments include but are not limited to Fv, Fab, Fab′,Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibodymolecules (e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The terms “anti-Ror2 antibody,” “Ror2 antibody” and “an antibody thatbinds to Ror2” as used herein refer to an antibody that is capable ofbinding Ror2 with sufficient affinity such that the antibody is usefulas a diagnostic and/or therapeutic agent in targeting ROr2. In oneembodiment, the extent of binding of an anti-Ror2 antibody to anunrelated, non-Ror2 protein is less than about 10% of the binding of theantibody to Ror2 as measured, e.g., by a radioimmunoassay (RIA). Incertain embodiments, an antibody that binds to Ror2 has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M). In certain embodiments, an anti-Ror2 antibody bindsto an epitope of Ror2 that is conserved among Ror2 from differentspecies.

The term “binding” as used herein refers to interaction of the variableregion or an Fv of an antibody with an antigen with the interactiondepending upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the antigen. For example, anantibody variable region or Fv recognizes and binds to a specificprotein structure rather than to proteins generally. As used herein, theterm “specifically binding” or “binding specifically” means that anantibody variable region or Fv binds to or associates with morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular antigen than with other proteins. Forexample, an antibody variable region or Fv specifically binds to itsantigen with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other antigens. For another example,an antibody variable region or Fv binds to a cell surface protein(antigen) with materially greater affinity than it does to relatedproteins or other cell surface proteins or to antigens commonlyrecognized by polyreactive natural antibodies (i.e., by naturallyoccurring antibodies known to bind a variety of antigens naturally foundin humans). However, “specifically binding” does not necessarily requireexclusive binding or non-detectable binding of another antigen, this ismeant by the term “selective binding”. In one example, “specificbinding” of an antibody variable region or Fv (or other binding region)binds to an antigen, means that the an antibody variable region or Fvbinds to the antigen with an equilibrium constant (KD) of 100 nM orless, such as 50 nM or less, for example 20 nM or less, such as, 15 nMor less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM orless.

The terms “cancer” and “cancerous” as used herein refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth/proliferation. Examples of cancer include,but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

The terms “cell proliferative disorder” and “proliferative disorder” asused herein refer to disorders that are associated with some degree ofabnormal cell proliferation. In one embodiment, the cell proliferativedisorder is cancer.

The term “chemotherapeutic agent” as used herein refers to a chemicalcompound useful in the treatment of cancer. Examples of chemotherapeuticagents include alkylating agents such as thiotepa and cyclosphosphamide(CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegall (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMF®); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®,Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib),proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors (see definition below);serine-threonine kinase inhibitors such as rapamycin (sirolimus,RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,SARASAR™); and pharmaceutically acceptable salts, acids or derivativesof any of the above; as well as combinations of two or more of the abovesuch as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics,” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and tripterelin; sex steroids, including progestines such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretionic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

The term “chimeric” antibody as used herein refers to an antibody inwhich a portion of the heavy and/or light chain is derived from aparticular source or species, while the remainder of the heavy and/orlight chain is derived from a different source or species.

The term “class” of an antibody as used herein refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “conditionally active antibody” as used herein refers to anantibody which is more active under a condition in the tumormicroenvironment compared to under a condition in the non-tumormicroenvironment. The conditions in the tumor microenvironment includelower pH, higher concentrations of lactate and pyruvate, hypoxia, lowerconcentration of glucose, and slightly higher temperature in comparisonwith non-tumor microenvironment. For example, a conditionally activeantibody is virtually inactive at normal body temperature, but is activeat a higher temperature in a tumor microenvironment. In yet anotheraspect, the conditionally active antibody is less active in normaloxygenated blood, but more active under a less oxygenated environmentexists in tumor. In yet another aspect, the conditionally activeantibody is less active in normal physiological pH 7.2-7.8, but moreactive under an acidic pH 5.8-7.0, or 6.0-6.8 that exists in a tumormicroenvironment. There are other conditions in the tumormicroenvironment know to a person skilled in the field may also be usedas the condition in the present invention under which the anti-Ror2antibodies to have different binding affinity to Ror2.

The term “constitutive” as used herein, as for example applied to Ror2activity, refers to continuous signaling activity of the receptor kinasethat is not dependent on the presence of a ligand or other activatingmolecules. Depending on the nature of the receptor kinase, all of theactivity may be constitutive or the activity of the receptor may befurther activated by the binding of other molecules (e.g. ligands).Cellular events that lead to activation of receptor kinase are wellknown among those of ordinary skill in the art. For example, activationmay include oligomerization, e.g., dimerization, trimerization, etc.,into higher order receptor complexes. Complexes may comprise a singlespecies of protein, i.e., a homomeric complex. Alternatively, complexesmay comprise at least two different protein species, i.e., a heteromericcomplex. Complex formation may be caused by, for example, overexpressionof normal or mutant forms of receptor on the surface of a cell. Complexformation may also be caused by a specific mutation or mutations in areceptor.

The term “cytostatic agent” as used herein refers to a compound orcomposition which arrests growth of a cell either in vitro or in vivo.Thus, a cytostatic agent may be one which significantly reduces thepercentage of cells in S phase. Further examples of cytostatic agentsinclude agents that block cell cycle progression by inducing G0/G1arrest or M-phase arrest. The humanized anti-Her2 antibody trastuzumab(HERCEPTIN®) is an example of a cytostatic agent that induces G0/G1arrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Certainagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in Mendelsohn and Israel, eds., TheMolecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W.B. Saunders,Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel)are anticancer drugs both derived from the yew tree. Docetaxel(TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is asemisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb).Paclitaxel and docetaxel promote the assembly of microtubules fromtubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited toradioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

The term “diabodies” as used herein refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites.

The term “detectably label” as used herein refers to any substance whosedetection or measurement, either directly or indirectly, by physical orchemical means, is indicative of the presence of the CTCs in a sample.Representative examples of useful detectable labels, include, but arenot limited to the following: molecules or ions directly or indirectlydetectable based on light absorbance, fluorescence, reflectance, lightscatter, phosphorescence, or luminescence properties; molecules or ionsdetectable by their radioactive properties; molecules or ions detectableby their nuclear magnetic resonance or paramagnetic properties. Includedamong the group of molecules indirectly detectable based on lightabsorbance or fluorescence, for example, are various enzymes which causeappropriate substrates to convert, e.g., from non-light absorbing tolight absorbing molecules, or from non-fluorescent to fluorescentmolecules.

The term “diagnostics” as used herein refers to determination of asubject's susceptibility to a disease or disorder, determination as towhether a subject is presently affected by a disease or disorder,prognosis of a subject affected by a disease or disorder (e. g.,identification of pre-metastatic or metastatic cancerous states, stagesof cancer, or responsiveness of cancer to therapy), and therametrics (e.g., monitoring a subject's condition to provide information as to theeffect or efficacy of therapy). In some embodiments, the diagnosticmethod of this invention is particularly useful in detecting early stagecancers.

The term “diagnostic agent” as used herein refers to a molecule whichcan be directly or indirectly detected and is used for diagnosticpurposes. The diagnostic agent may be administered to a subject or asample. The diagnostic agent can be provided per se or may be conjugatedto a vehicle such as a conditionally active antibody.

The term “effector functions” as used herein refer to those biologicalactivities attributable to the Fc region of an antibody, which vary withthe antibody isotype. Examples of antibody effector functions include:C1q binding and complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g. B cellreceptor); and B cell activation.

The term “effective amount” of an agent as used herein, e.g., apharmaceutical formulation, refers to an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result.

The term “Fc region” as used herein is used to define a C-terminalregion of an immunoglobulin heavy chain that contains at least a portionof the constant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

The term “framework” or “FR” as used herein refers to variable domainresidues other than hypervariable region (HVR or H1-3 in the heavy chainand L1-3 in the light chain) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in V_(H) (or V_(L)): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “full length antibody,” “intact antibody,” or “whole antibody”refers to an antibody which comprises an antigen-binding variable region(V_(H) or V_(L)) as well as a light chain constant domain (CL) and heavychain constant domains, CH1, CH2 and CH3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variants thereof. Depending on the aminoacid sequence of the constant domain of their heavy chains, full lengthantibodies can be assigned to different “classes”. There are five majorclasses of full length antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into “subclasses” (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constantdomains that correspond to the different classes of antibodies arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The terms “host cell,” “host cell line,” and “host cell culture” as usedherein are used interchangeably and refer to cells into which exogenousnucleic acid has been introduced, including the progeny of such cells.Host cells include “transformants” and “transformed cells,” whichinclude the primary transformed cell and progeny derived therefromwithout regard to the number of passages. Progeny may not be completelyidentical in nucleic acid content to a parent cell, but may containmutations. Mutant progeny that have the same function or biologicalactivity as screened or selected for in the originally transformed cellare included herein.

The term “human antibody” as used herein is one which possesses an aminoacid sequence which corresponds to that of an antibody produced by ahuman or a human cell or derived from a non-human source that utilizeshuman antibody repertoires or other human antibody-encoding sequences.This definition of a human antibody specifically excludes a humanizedantibody comprising non-human antigen-binding residues.

The term “human consensus framework” as used herein is a framework whichrepresents the most commonly occurring amino acid residues in aselection of human immunoglobulin V_(L) or V_(H) framework sequences.Generally, the selection of human immunoglobulin V_(L) or V_(H)sequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for theV_(L), the subgroup is subgroup kappa I as in Kabat et al., supra. Inone embodiment, for the V_(H), the subgroup is subgroup III as in Kabatet al., supra.

The term “humanized” antibody as used herein refers to a chimericantibody comprising amino acid residues from non-human HVRs and aminoacid residues from human FRs. In certain embodiments, a humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the HVRs(e.g., CDRs) correspond to those of a non-human antibody, and all orsubstantially all of the FRs correspond to those of a human antibody. Ahumanized antibody optionally may comprise at least a portion of anantibody constant region derived from a human antibody. A “humanizedform” of an antibody, e.g., a non-human antibody, refers to an antibodythat has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theV_(H) (H1, H2, H3), and three in the V_(L) (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol., vol. 196, pp. 901-917 1987) ExemplaryCDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at aminoacid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. 1991). With the exception of CDR1 inV_(H), CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008). Unlessotherwise indicated, HVR residues and other residues in the variabledomain (e.g., FR residues) are numbered herein according to Kabat etal., supra.

The term “immunoconjugate” as used herein is an antibody conjugated toone or more heterologous molecule(s), including but not limited to acytotoxic agent.

The term “individual” or “subject” as used herein refers to a mammal.Mammals include, but are not limited to, domesticated animals (e.g.,cows, sheep, cats, dogs, and horses), primates (e.g., humans andnon-human primates such as monkeys), rabbits, and rodents (e.g., miceand rats). In certain embodiments, the individual or subject is a human.

The term “inhibiting cell growth or proliferation” as used herein meansdecreasing a cell's growth or proliferation by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing celldeath.

The term “isolated” antibody as used herein is one which has beenseparated from a component of its natural environment. In someembodiments, an antibody is purified to greater than 95% or 99% purityas determined by, for example, electrophoretic (e.g., SDS-PAGE,isoelectric focusing (IEF), capillary electrophoresis) orchromatographic (e.g., ion exchange or reverse phase High PerformanceLiquid Chromatography (HPLC)). For review of methods for assessment ofantibody purity, see, e.g., Flatman et al., J. Chromatogr. B, vol. 848,pp. 79-87, 2007.

The term “isolated” nucleic acid as used herein refers to a nucleic acidmolecule that has been separated from a component of its naturalenvironment. An isolated nucleic acid includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

The term “isolated nucleic acid encoding an anti-Ror2 antibody” as usedherein refers to one or more nucleic acid molecules encoding antibodyheavy and light chains (or fragments thereof), including such nucleicacid molecule(s) in a single vector or separate vectors, and suchnucleic acid molecule(s) present at one or more locations in a hostcell.

The term “ligand-independent” as used herein, as for example applied toreceptor signaling activity, refers to signaling activity that is notdependent on the presence of a ligand. A receptor havingligand-independent kinase activity will not necessarily preclude thebinding of ligand to that receptor to produce additional activation ofthe kinase activity.

The term “metastasis” as used herein refers to all Ror2-involvingprocesses that support cancer cells to disperse from a primary tumor,penetrate into lymphatic and/or blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasis) in normal tissueselsewhere in the body. In particular, it refers to cellular events oftumor cells such as proliferation, migration, anchorage independence,evasion of apoptosis, or secretion of angiogenic factors, that underliemetastasis and are stimulated or mediated by non-catalytic or catalyticactivities of Ror2, preferably including Ror2 phosphorylation and/orRor2-mediated signal transduction.

The term “microenvironment” as used herein means any portion or regionof a tissue or body that has constant or temporal, physical or chemicaldifferences from other regions of the tissue or regions of the body. Fortumors, the term “tumor microenvironment” as used herein refers to theenvironment in which a tumor exists, which is the non-cellular areawithin the tumor and the area directly outside the tumorous tissue butdoes not pertain to the intracellular compartment of the cancer cellitself. The tumor and the tumor microenvironment are closely related andinteract constantly. A tumor can change its microenvironment, and themicroenvironment can affect how a tumor grows and spreads. Typically,the tumor microenvironment has a low pH in the range of 5.8 to 7.0, morecommonly in the range of 6.0 to 6.8, in the range of 6.2-6.8. On theother hand, a normal physiological pH is in the range of 7.2-7.8. Thetumor microenvironment is also known to have lower concentration ofglucose and other nutrients, but higher concentration of lactic acid, incomparison with blood plasma. Furthermore, the tumor microenvironmentcan have a temperature that is 0.3 to 1° C. higher than the normalphysiological temperature. The tumor microenvironment has been discussedin Gillies et al., “MRI of the Tumor Microenvironment,” Journal ofMagnetic Resonance Imaging, vol. 16, pp. 430-450, 2002, herebyincorporated by reference herein its entirety. The term “non-tumormicroenvironment” refers to a microenvironment at a site other than atumor.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “naked antibody” as used herein refers to an antibody that isnot conjugated to a heterologous moiety (e.g., a cytotoxic moiety) orradiolabel. The naked antibody may be present in a pharmaceuticalformulation.

The term “native antibodies” as used herein refers to naturallyoccurring immunoglobulin molecules with varying structures. For example,native IgG antibodies are heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light chains and twoidentical heavy chains that are disulfide-bonded. From N- to C-terminus,each heavy chain has a variable region (V_(H)), also called a variableheavy domain or a heavy chain variable domain, followed by threeconstant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus,each light chain has a variable region (V_(L)), also called a variablelight domain or a light chain variable domain, followed by a constantlight (C_(L)) domain. The light chain of an antibody may be assigned toone of two types, called kappa (κ) and lambda (2), based on the aminoacid sequence of its constant domain.

The term “package insert” as used herein is used to refer toinstructions customarily included in commercial packages of therapeuticproducts, that contain information about the indications, usage, dosage,administration, combination therapy, contraindications and/or warningsconcerning the use of such therapeutic products.

The term “percent (%) amino acid sequence identity” with respect to areference polypeptide sequence as used herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the reference polypeptidesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters foraligning sequences, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Forpurposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” as used herein refers to apreparation which is in such form as to permit the biological activityof an active ingredient contained therein to be effective, and whichcontains no additional components which are unacceptably toxic to asubject to which the formulation would be administered.

The term “pharmaceutically acceptable carrier” as used herein refers toan ingredient in a pharmaceutical formulation, other than an activeingredient, which is nontoxic to a subject., A pharmaceuticallyacceptable carrier includes, but is not limited to, a buffer, excipient,stabilizer, or preservative.

The terms “purified” and “isolated” used herein refer to an antibodyaccording to the invention or to a nucleotide sequence, that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. The term “purified” as usedherein preferably means at least 75% by weight, more preferably at least85% by weight, more preferably still at least 95% by weight, and mostpreferably at least 98% by weight, of biological macromolecules of thesame type are present. An “isolated” nucleic acid molecule which encodesa particular polypeptide refers to a nucleic acid molecule which issubstantially free of other nucleic acid molecules that do not encodethe polypeptide; however, the molecule may include some additional basesor moieties which do not deleteriously affect the basic characteristicsof the composition.

The term “recombinant antibody” as used herein refers to an antibody(e.g. a chimeric, humanized, or human antibody or antigen-bindingfragment thereof) that is expressed by a recombinant host cellcomprising nucleic acid encoding the antibody. Examples of “host cells”for producing recombinant antibodies include: (1) mammalian cells, forexample, Chinese Hamster Ovary (CHO), COS, myeloma cells (including Y0and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2)insect cells, for example, sf9, sf21 and Tn5; (3) plant cells, forexample plants belonging to the genus Nicotiana (e.g. Nicotianatabacum); (4) yeast cells, for example, those belonging to the genusSaccharomyces (e.g. Saccharomyces cerevisiae) or the genus Aspergillus(e.g. Aspergillus niger); (5) bacterial cells, for example Escherichia.coli cells or Bacillus subtilis cells, etc.

The term “Ror2” as used herein, refers to receptor tyrosine kinase-likeorphan receptor 2, which is a predicted 943-amino acid protein with invitro protein kinase activity, shown in Genbank accession numberAAI30523. Many lineage-restricted receptor tyrosine kinases wereinitially identified as ‘orphans’ homologous to known receptors, andonly subsequently used to identify their unknown growth factors.DeChiara et al. (2000) identified one such orphan, encoded by Ror2 asshown in FIG. 1.

The term “therapeutically effective amount” of the antibody of theinvention is meant a sufficient amount of the antibody to treat saidcancer, at a reasonable benefit/risk ratio applicable to any medicaltreatment. It will be understood, however, that the total daily usage ofthe antibodies and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificantibody employed; the specific composition employed, the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific antibody employed; the duration of the treatment; drugs used incombination or coincidental with the specific antibody employed; andlike factors well known in the medical arts. For example, it is wellknown within the skill of the art to start doses of the compound atlevels lower than those required to achieve the desired therapeuticeffect and to gradually increase the dosage until the desired effect isachieved.

The term “single chain Fv” (“scFv”) as used herein is a covalentlylinked V_(H)::V_(L) heterodimer which is usually expressed from a genefusion including V_(H) and V_(L) encoding genes linked by apeptide-encoding linker. “dsFv” is a V_(H)::V_(L) heterodimer stabilisedby a disulfide bond. Divalent and multivalent antibody fragments canform either spontaneously by association of monovalent scFvs, or can begenerated by coupling monovalent scFvs by a peptide linker, such asdivalent sc(Fv)2.

The term “treatment,” “treat,” or “treating” as used herein refers toclinical intervention in an attempt to alter the natural course of theindividual being treated, and can be performed either for prophylaxis orduring the course of clinical pathology. Desirable effects of treatmentinclude, but are not limited to, preventing occurrence or recurrence ofdisease, alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, preventing metastasis,decreasing the rate of disease progression, amelioration or palliationof the disease state, and remission or improved prognosis. In someembodiments, antibodies of the invention are used to delay developmentof a disease or to slow the progression of a disease.

The term “tumor” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The term “variable region” or “variable domain” as used herein refers tothe domain of an antibody heavy or light chain that is involved inbinding the antibody to antigen. The variable domains of the heavy chainand light chain (V_(H) and V_(L), respectively) of a native antibodygenerally have similar structures, with each domain comprising fourconserved framework regions (FRs) and three hypervariable regions(HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freemanand Co., page 91 (2007).) A single V_(H) or V_(L) domain may besufficient to confer antigen-binding specificity. Furthermore,antibodies that bind a particular antigen may be isolated using a V_(H)or V_(L) domain from an antibody that binds the antigen to screen alibrary of complementary V_(L) or V_(H) domains, respectively. See,e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993;Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.

The term “vector” as used herein refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

DETAILED DESCRIPTION

For illustrative purposes, the principles of the present invention aredescribed by referencing various exemplary embodiments. Although certainembodiments of the invention are specifically described herein, one ofordinary skill in the art will readily recognize that the sameprinciples are equally applicable to, and can be employed in, othersystems and methods. Before explaining the disclosed embodiments of thepresent invention in detail, it is to be understood that the inventionis not limited in its application to the details of any particularembodiment shown. Additionally, the terminology used herein is for thepurpose of description and not for limitation. Furthermore, althoughcertain methods are described with reference to steps that are presentedherein in a certain order, in many instances, these steps can beperformed in any order as may be appreciated by one skilled in the art;the novel method is therefore not limited to the particular arrangementof steps disclosed herein.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. Furthermore, the terms “a” (or“an”), “one or more”, and “at least one” can be used interchangeablyherein. The terms “comprising”, “including”, “having” and “constructedfrom” can also be used interchangeably.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, percent, ratio,reaction conditions, and so forth used in the specification and claimsare to be understood as being modified in all instances by the term“about,” whether or not the term “about” is present. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thespecification and claims are approximations that may vary depending uponthe desired properties sought to be obtained by the present disclosure.At the very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the disclosure are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

It is to be understood that each component, compound, substituent, orparameter disclosed herein is to be interpreted as being disclosed foruse alone or in combination with one or more of each and every othercomponent, compound, substituent, or parameter disclosed herein.

It is also to be understood that each amount/value or range ofamounts/values for each component, compound, substituent, or parameterdisclosed herein is to be interpreted as also being disclosed incombination with each amount/value or range of amounts/values disclosedfor any other component(s), compounds(s), substituent(s), orparameter(s) disclosed herein and that any combination of amounts/valuesor ranges of amounts/values for two or more component(s), compounds(s),substituent(s), or parameters disclosed herein are thus also disclosedin combination with each other for the purposes of this description.

It is further understood that each lower limit of each range disclosedherein is to be interpreted as disclosed in combination with each upperlimit of each range disclosed herein for the same component, compounds,substituent, or parameter. Thus, a disclosure of two ranges is to beinterpreted as a disclosure of four ranges derived by combining eachlower limit of each range with each upper limit of each range. Adisclosure of three ranges is to be interpreted as a disclosure of nineranges derived by combining each lower limit of each range with eachupper limit of each range, etc. Furthermore, specific amounts/values ofa component, compound, substituent, or parameter disclosed in thedescription or an example is to be interpreted as a disclosure of eithera lower or an upper limit of a range and thus can be combined with anyother lower or upper limit of a range or specific amount/value for thesame component, compound, substituent, or parameter disclosed elsewherein the application to form a range for that component, compound,substituent, or parameter.

A. Anti-Ror2 Antibodies

In one aspect, the present invention provides an isolated heavy chainvariable region polypeptide that specifically binds to human Ror2protein. The heavy chain variable region polypeptide comprises threecomplementarity determining regions H1, H2, and H3 sequences, wherein:

-   -   the H1 sequence is GYTX₁TEX₂X₃X₄H (SEQ ID NO:1) or GYSITTGX₂₉YWN        (SEQ ID NO:4);    -   the H2 sequence is X₅X₆X₇X₈NNGGTGYNQKFKG (SEQ ID NO:2) or        YITYDGSX₃₀NYNPSLKN (SEQ ID NO:5); and    -   the H3 sequence is X₉X₁₀X₁₁SX₁₂YX₁₃YX₁₄X₁₅SYFX₁₆X₁₇X₁₈ (SEQ ID        NO:3) or CSX₃₁X₃₂X₃₃X₃₄VX₃₅X₃₆X₃₇LDX₃₈ (SEQ ID NO:6);

wherein

X₁ is F or E,

X₂ is Y or D,

X₃ is T or C,

X₄ is M or D or E or Y,

X₅ is G or S,

X₆ is I or E,

X₇ is N or C or L or V,

X₈ is T or D or E,

X₉ is A or M or T,

X₁₀ is R or H,

X₁₁ is G or E,

X₁₂ is L or F,

X₁₃ is S or G,

X₁₄ is G or D,

X₁₅ is N or E,

X₁₆ is D or L,

X₁₇ is Y or C or T,

X₁₈ is W or L,

X₂₉ is Y or E or R or T,

X₃₀ is K or N,

X₃₁ is R or G or H or W or Y,

X₃₂ is F or C or N or Q,

X₃₃ is E or S,

X₃₄ is G or E or F or H or M or Q or S,

X₃₅ is W or A or I or P or Q or T or V,

X₃₆ is Y or G or N or Q,

X₃₇ is G or S or T, and

X₃₈ is Y or I.

The alignment of the heavy chain variable regions is shown in FIGS.2A-2B.

In another aspect, the present invention provides an isolated lightchain variable region polypeptide that specifically binds to human Ror2protein. The light chain variable region polypeptide comprises threecomplementarity determining regions L1, L2, and L3 sequences, wherein:

-   -   the L1 sequence is SATSSX₁₉X₂₉X₂₁MX₂₂ (SEQ ID NO:7) or        RASESVDRYGNSX₃₉IH (SEQ ID NO:10);    -   the L2 sequence is X₂₃TSNLAS (SEQ ID NO:8) or X₄₀TYX₄₁LES (SEQ        ID NO:11); and    -   the L3 sequence is QX₂₄X₂₅SX₂₆YPFX₂₇X₂₈ (SEQ ID NO:9) or        QQX₄₂NX₄₃DPX₄₄TX₄₅ (SEQ ID NO:12);

wherein

X₁₉ is V or E,

X₂₀ is S or D,

X₂₁ is Y or C or D,

X₂₂ is H or G or L,

X₂₃ is G or C or H or P,

X₂₄ is Q or E,

X₂₅ is R or H,

X₂₆ is S or D or G or I or Q or V,

X₂₇ is T or D,

X₂₈ is F or D or E,

X₃₉ is F or S or T,

X₄₀ is R or C or D or E or W,

X₄₁ is N or D,

X₄₂ is T or I or P,

X₄₃ is E or V,

X₄₄ is W or T, and

X₄₅ is F or T.

The alignment of the light chain variable regions is shown in FIGS.3A-3B.

The present invention identified these isolated heavy chain variableregions and isolated light chain variable regions, respectively, fromthe heavy chain variable region and light chain variable region of aparent antibody through a method disclosed in U.S. Pat. No. 8,709,755.This method of generating a conditionally active antibody is herebyincorporated by reference herein.

The DNAs encoding the heavy chain variable region and light chainvariable region the parent antibody were evolved to generate mutantantibody libraries using Comprehensive Positional Evolution (CPE), whicheach position in the heavy chain variable region and light chainvariable region of the parent antibody is randomized one at a time. Eachmutant heavy chain/light chain in the libraries has only one singlepoint mutation, in comparison with the heavy chain variable region orlight chain variable region of the parent antibody (FIGS. 2A-2B and3A-3B). The mutants in the libraries were screened for selective bindingaffinity to human Ror2 at pH 6.0 over pH 7.4 by ELISA. The mutant heavychain/light chain variable regions that are more active at pH 6.0 thanat pH 7.4 were selected as the heavy chain/light chain variable regionsof conditionally active antibodies, with the single point mutationsindicated in each of the heavy chain and light chain variable region(Tables 1 and 2, FIGS. 2A-2B and 3A-3B).

TABLE 1 Conditionally active anti-Ror2 antibody light chain variableregions Affinity ELISA Mutants ratio, pH Clone pH 6.0 pH 7.4 6.0/7.4LC-V029E 0.406 0.129 3.15 LC-S030D 0.733 0.252 2.91 LC-Y031C 1.341 0.7001.92 LC-Y031D 0.911 0.405 2.25 LC-H033G 1.103 0.536 2.06 LC-H033L 0.6900.332 2.08 LC-G049C 0.468 0.133 3.52 LC-G049H 1.873 0.118 15.87 LC-G049P0.660 0.268 2.46 LC-Q089E 0.438 0.251 1.74 LC-R090H 0.560 0.256 2.19LC-S092D 1.127 1.122 1.00 LC-S092V 1.226 1.182 1.04 LC-T096D 0.755 0.4741.59 LC-F097D 0.190 0.107 1.77 LC-F097E 0.489 0.215 2.28

TABLE 2 Conditionally active anti-Ror2 antibody heavy chain variableregions Affinity ELISA mutants ratio, pH Clone pH 6.0 pH 7.4 6.0/7.4HC-F029E 1.342 0.742 1.81 HC-Y032D 1.470 0.510 2.88 HC-T033C 0.626 0.2322.70 HC-M034D 1.244 0.211 5.90 HC-M034E 1.165 0.185 6.30 HC-M034Y 0.9030.391 2.31 HC-G050S 0.823 0.318 2.60 HC-I051E 1.039 0.100 10.39 HC-N052C0.995 0.431 2.31 HC-N052L 0.911 0.433 2.10 HC-N052V 0.870 0.340 2.56HC-T053D 1.108 0.222 4.99 HC-T053E 0.540 0.157 3.44 HC-A097I 1.892 1.7301.892 HC-A097M 1.977 1.701 1.977 HC-A097S 1.666 1.692 1.666 HC-A097T1.834 1.692 1.666 HC-R098H 0.928 0.353 2.63 HC-R098Q 0.799 0.276 2.89HC-G099E 0.616 0.421 1.46 HC-L101F 1.461 1.412 1.461 HC-S103G 0.7940.655 1.21 HC-G105D 0.509 0.379 1.34 HC-N106E 0.224 0.115 1.95 HC-D110L1.675 1.403 1.675 HC-Y111C 1.079 0.793 1.36 HC-Y111T 1.862 1.403 1.675HC-W112L 1.746 1.704 1.746

In another aspect, the present invention includes the heavy chainvariable regions as represented in FIGS. 2A-2B and the light chainvariable regions as presented in FIGS. 3A-3B. Amino acid sequences ofthe heavy chain variable regions are SEQ ID NOS: 18-26. The amino acidsequences of the light chain variable regions are SEQ ID NOS: 13-17 and27. These heavy chain variable regions and light chain variable regionscan specifically bind to human Ror2. Antibodies or antibody fragmentscomprising one of these heavy chain variable regions and light chainvariable regions have been found to have higher binding affinity to Ror2at a pH in the tumor microenvironment than at a pH in a non-tumormicroenvironment. For example, the antibodies and antibody fragmentshave a higher binding affinity to Ror2 at pH 6.0 than at pH 7.4.

The anti-Ror2 antibodies or antibody fragments have a higher bindingaffinity to Ror2 in a tumor in comparison with their binding affinity toRor2 in a normal tissue. These anti-Ror2 antibodies or antibodyfragments have a longer half-life and reduced side-effects, as well ascomparable efficacy, in comparison with monoclonal anti-Ror2 antibodiesknown in the art. These features permit use of a higher dosage of theseanti-Ror2 antibodies or antibody fragments to be delivered to a patientthus being a more effective therapeutic option.

Though the present invention includes the heavy chain variable regionsand light chain variable regions presented in FIGS. 2A-2B, 3A-3B andhaving amino acid sequences with SEQ ID NOS: 13-24, the presentinvention also provides variants thereof that can specifically bind tohuman Ror2. In order to derive these variants, the complementaritydetermining regions (CDRs) of the heavy chain variable regions (H1-H3)and the complementarity determining regions of the light chain variableregions (L1-L3) should remain intact. However, the amino acid sequenceof the heavy chain variable regions and light chains variable regionsoutside of the complementarity determining regions may be mutated inaccordance with the principles of substitution, insertion and deletionas discussed in this application.

In deriving these variants, one is guided by the process as describedherein. The variants of the heavy chain variable regions and light chainvariable regions may be prepared by introducing appropriatemodifications into the nucleotide sequence encoding the heavy chainvariable regions and light chain variable regions, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of residues within the aminoacid sequences of the heavy chain variable regions and light chainvariable regions. Any combination of deletion, insertion, andsubstitution can be made to arrive at the antibodies or antibodyfragments of the present invention, provided that they possess thedesired characteristics, e.g., antigen-binding to human Ror2 and/orconditional activity.

Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody or antibody fragment variants havingone or more amino acid substitutions are provided. Sites of interest forsubstitutional mutagenesis include the CDRs and framework regions (FRs).Conservative substitutions are shown in Table 3 under the heading of“conservative substitutions.” More substantial changes are provided inTable 3 under the heading of “exemplary substitutions,” and as furtherdescribed below in reference to amino acid side chain classes. Aminoacid substitutions may be introduced into an antibody or antibodyfragment of interest and the products screened for a desired activity,e.g., retained/improved antigen binding, or decreased immunogenicity.

TABLE 3 Amino acid substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore CDR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in CDRs, e.g., to improveantibody affinity. Such alterations may be made in CDR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol., vol. 207, pp. 179-196, 2008), and/or SDRs (a-CDRs), with theresulting variant VH or VL being tested for binding affinity. Affinitymaturation by constructing and reselecting from secondary libraries hasbeen described, e.g., in Hoogenboom et al. in Methods in MolecularBiology, vol. 178, pp. 1-37, 2001). In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves CDR-directed approaches, in which several CDRresidues (e.g., 4-6 residues at a time) are randomized. CDR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody or antibody fragment tobind antigen. For example, conservative alterations (e.g., conservativesubstitutions as provided herein) that do not substantially reducebinding affinity may be made in CDRs. Such alterations may be outside ofCDR “hotspots” or SDRs. In certain embodiments of the variant V_(H) andV_(L) sequences provided above, each CDR either is unaltered, orcontains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells, Science, vol. 244,pp. 1081-1085, 1989. In this method, a residue or group of targetresidues (e.g., charged residues such as arg, asp, his, lys, and glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody or antibody fragment with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antibody complex to identify contact points between the antibodyor antibody fragment and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody include the fusion to the N- or C-terminus ofthe antibody to an enzyme (e.g. for ADEPT) or a polypeptide whichincreases the serum half-life of the antibody.

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody. Itis known that when a humanized antibody is produced by simply graftingonly CDRs in V_(H) and V_(L) of an antibody derived from a non-humananimal in FRs of the V_(H) and V_(L) of a human antibody, the antigenbinding activity is reduced in comparison with that of the originalantibody derived from a non-human animal. It is considered that severalamino acid residues of the V_(H) and V_(L) of the non-human antibody,not only in CDRs but also in FRs, are directly or indirectly associatedwith the antigen binding activity. Hence, substitution of these aminoacid residues with different amino acid residues derived from FRs of theV_(H) and V_(L) of the human antibody would reduce of the bindingactivity. In order to resolve the problem, in antibodies grafted withhuman CDR, attempts have to be made to identify, among amino acidsequences of the FR of the V_(H) and V_(L) of human antibodies, an aminoacid residue which is directly associated with binding to the antibody,or which interacts with an amino acid residue of CDR, or which maintainsthe three-dimensional structure of the antibody and which is directlyassociated with binding to the antigen. The reduced antigen bindingactivity could be increased by replacing the identified amino acids withamino acid residues of the original antibody derived from a non-humananimal.

Modifications and changes may be made in the structure of the antibodiesof the present invention, and in the DNA sequences encoding them, andstill obtain a functional molecule that encodes an antibody withdesirable characteristics.

In making the changes in the amino sequences, the hydropathic index ofamino acids may be considered. The importance of the hydropathic aminoacid index in conferring interactive biologic function on a protein isgenerally understood in the art. It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine(+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine(−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline(−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate(−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

A further object of the present invention also encompassesfunction-conservative variants of the antibodies of the presentinvention.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids otherthan those indicated as conserved may differ in a protein so that thepercent protein or amino acid sequence similarity between any twoproteins of similar function may vary and may be, for example, from 70%to 99% as determined according to an alignment scheme such as by theCluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function-conservative variant” also includes a polypeptide which has atleast 60% amino acid identity as determined by BLAST or FASTAalgorithms, preferably at least 75%, more preferably at least 85%, stillpreferably at least 90%, and even more preferably at least 95%, andwhich has the same or substantially similar properties or functions asthe native or parent protein to which it is compared.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than 80%, preferably greater than85%, preferably greater than 90% of the amino acids are identical, orgreater than about 90%, preferably greater than 95%, are similar(functionally identical) over the whole length of the shorter sequence.Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program, orany of sequence comparison algorithms such as BLAST, FASTA, etc.

For example, certain amino acids may be substituted by other amino acidsin a protein structure without appreciable loss of activity. Since theinteractive capacity and nature of a protein define the protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and, of course, in its DNA encodingsequence, while nevertheless obtaining a protein with like properties.It is thus contemplated that various changes may be made in thesequences of the antibodies or antibody fragments of the invention, orcorresponding DNA sequences which encode said antibodies or antibodyfragments, without appreciable loss of their biological activity.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH, vol. 15, pp. 26-32, 1997. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol., vol. 336, pp. 1239-1249, 2004; Yamane-Ohnuki et al.Biotech. Bioeng., vol. 87, pp. 614-622, 2004. Examples of cell linescapable of producing defucosylated antibodies include Lec13 CHO cellsdeficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.,vol. 249, pp. 533-545, 1986; US Pat Appl No US 2003/0157108 A; and WO2004/056312 A1, especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004;Kanda, Y. et al., Biotechnol. Bioeng., vol. 94, pp. 680-688, 2006; andWO2003/085107).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

Fe Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such asADCC) are unnecessary or deleterious. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγR1, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991. Non-limitingexamples of in vitro assays to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 (see also, e.g.Hellstrom et al. Proc. Nat'l Acad. Sci. USA, vol. 83, pp. 7059-7063,1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp.1499-1502, 1985; U.S. Pat. No. 5,821,337 (see also Bruggemann et al., J.Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicityassay (Promega, Madison, Wis.). Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol.95, pp. 652-656, 1998. C1q binding assays may also be carried out toconfirm that the antibody is unable to bind C1q and hence lacks CDCactivity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J. Immunol.Methods, vol. 202, pp. 163-171, 1996; Cragg, M. S. et al., Blood, vol.101, pp. 1045-1052, 2003; and Cragg, M. S, and M. J. Glennie, Blood,vol. 103, pp. 2738-2743, 2004). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l Immunol., vol. 18, pp.1759-1769, 2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem., vol. 9, pp. 6591-6604, 2001).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol., vol. 164,pp. 4178-4184, 2000.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol., vol. 117, pp.587-593, 1976 and Kim et al., J. Immunol., vol. 24, p. 249, 1994), aredescribed in US2005/0014934. Those antibodies comprise an Fc region withone or more substitutions therein which improve binding of the Fc regionto FcRn. Such Fc variants include/e those with substitutions at one ormore of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g.,substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). Seealso Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat.Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other examples ofFc region variants.

Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

Antibody Derivatives

In certain embodiments, an antibody or antibody fragment provided hereinmay be further modified to contain additional nonproteinaceous moietiesthat are known in the art and readily available. The moieties suitablefor derivatization of the antibody or antibody fragment include but arenot limited to water soluble polymers. Non-limiting examples of watersoluble polymers include, but are not limited to, polyethylene glycol(PEG), copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody or antibodyfragment may vary, and if more than one polymer are attached, they canbe the same or different molecules. In general, the number and/or typeof polymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody or antibody fragment to be improved,whether the derivative will be used in a therapy under definedconditions, etc.

In another embodiment, conjugates of an antibody or antibody fragmentand nonproteinaceous moiety that may be selectively heated by exposureto radiation are provided. In one embodiment, the nonproteinaceousmoiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA,vol. 102, pp. 11600-11605, 2005). The radiation may be of anywavelength, and includes, but is not limited to, wavelengths that do notharm ordinary cells, but which heat the nonproteinaceous moiety to atemperature at which cells proximal to the antibody-nonproteinaceousmoiety are killed.

In another aspect, the present invention provides an anti-Ror2 antibodyor antibody fragment including the isolated heavy chain variable regionpolypeptides or isolated light chain variable region polypeptides. Theisolated heavy chain variable region polypeptides comprise the H1, H2,and H3 regions with SEQ ID NOS: 1-6. The isolated light chain variableregion polypeptides comprise the L1, L2, and L3 regions with SEQ ID NOS:7-12.

The anti-Ror2 antibody or antibody fragment of the invention has ahigher binding affinity to Ror2 under a condition in tumormicroenvironment than under a condition in a non-tumor microenvironment.In one embodiment, the condition in tumor microenvironment and thecondition in a non-tumor microenvironment are both pH. The anti-Ror2antibodies or antibody fragments of the invention thus can selectivelybind to Ror2 at a pH about 5.of-6.8 but will have a lower bindingaffinity to Ror2 at a pH about 7.2-7.8 encountered in a normalphysiological environment. As shown Example 1, the anti-Ror2 antibodiesor antibody fragments have higher binding affinity to Ror2 at pH 6.0that at pH 7.4.

In certain embodiments, the anti-Ror2 antibodies or antibody fragmentsof the present invention have a dissociation constant (Kd) with Ror2under a condition in tumor microenvironment of about ≤1 μM, ≤100 nM, ≤10nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, or from10⁻⁸M to 10⁻¹³M, or from 10⁻⁹M to 10^(|13) M). In one embodiment, theratio of the Kd of the antibody or antibody fragment with Ror2 at avalue of the condition in tumor microenvironment to the Kd at adifferent value of the same condition in non-tumor microenvironment isat least about 1.5:1, at least about 2:1, at least about 3:1, at leastabout 4:1, at least about 5:1, at least about 6:1, at least about 7:1,at least about 8:1, at least about 9:1, at least about 10:1, at leastabout 20:1, at least about 30:1, at least about 50:1, at least about70:1, or at least about 100:1.

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen using the following assay. Solution binding affinity ofFabs for antigen is measured by equilibrating Fab with a minimalconcentration of (¹²⁵I)-labeled antigen in the presence of a titrationseries of unlabeled antigen, then capturing bound antigen with ananti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips atabout 10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml C0.204) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

The anti-Ror2 antibodies of the invention may be a chimeric, humanizedor human antibody. In one embodiment, an anti-Ror2 antibody fragment isemployed, e.g., a Fv, Fab, Fab′, Fab′-SH, scFv, a diabody, a triabody, atetrabody or an F(ab′)₂ fragment and multispecific antibodies formedfrom antibody fragments. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG antibody or other antibody class orisotype as defined herein. For a review of certain antibody fragments,see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For a review ofscFv fragments, see, e.g., Pluckthün, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, NewYork), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)₂ fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046.

The diabodies of the invention may be bivalent or bispecific. See, forexample, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134(2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp.6444-6448, 1993 for examples of diabodies. Examples of triabodies andtetrabodies are also described in Hudson et al., Nat. Med., vol. 9, pp.129-134, 2003.

In some embodiments, the invention comprises single-domain antibodyfragments comprising all or a portion of the heavy chain variable domainor all or a portion of the light chain variable domain of an antibody.In certain embodiments, a single-domain antibody is a humansingle-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S.Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

In some embodiments, the anti-Ror2 antibodies of the invention may bechimeric antibodies. Certain chimeric antibodies are described, e.g., inU.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.USA, vol. 81, pp. 6851-6855, 1984). In one example, the chimericantibody comprises a non-human variable region (e.g., a variable regionderived from a mouse, rat, hamster, rabbit, or non-human primate, suchas a monkey) and a human constant region. In a further example, thechimeric antibody is a “class switched” antibody in which the class orsubclass of the antibody has been changed relative to the class orsubclass of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof

In certain embodiments, the chimeric antibody of the invention is ahumanized antibody. Typically, such a non-human antibody is humanized toreduce immunogenicity to humans, while retaining the specificity andaffinity of the parental non-human antibody. Generally, a humanizedantibody comprises one or more variable domains in which CDRs (orportions thereof) are derived from a non-human antibody, and FRs (orportions thereof) are derived from human antibody sequences. A humanizedantibody may optionally also comprise at least a portion of a humanconstant region. In some embodiments, some FR residues in a humanizedantibody are substituted with corresponding residues from a non-humanantibody (e.g., the antibody from which the CDR residues are derived),e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, andare further described, e.g., in Riechmann et al., Nature, vol. 332, pp.323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA, vol. 86, pp.10029-10033, 1989; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri et al., Methods, vol. 36, pp. 25-34, 2005(describing SDR (a-CDR) grafting); Padlan, Mol. Immunol., vol. 28, pp.489-498, 1991 (describing “resurfacing”); Dall'Acqua et al., Methods,vol. 36, pp. 43-60, 2005 (describing “FR shuffling”); and Osbourn etal., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer,vol. 83, pp. 252-260, 2000 (describing the “guided selection” approachto FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993);framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89,p. 4285, 1992; and Presta et al. J. Immunol., vol. 151, p. 2623, 1993);human mature (somatically mutated) framework regions or human germlineframework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol.13, pp. 1619-1633, 2008); and framework regions derived from screeningFR libraries (see, e.g., Baca et al., J. Biol. Chem., vol. 272, pp.10678-10684, 1997 and Rosok et al., J. Biol. Chem., vol. 271, pp.22611-22618, 1996).

In some embodiments, the anti-Ror2 antibodies of the invention aremultispecific, e.g. bispecific antibodies. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for Ror2 and the other is for another antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of Ror2. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express Ror2. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature, vol. 305, pp. 537-540, 1983), WO 93/08829, andTraunecker et al., EMBO J. vol. 10, pp. 3655-3659, 1991), and“knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168).Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, vol. 229,pp. 81-83, 1985); using leucine zippers to produce bi-specificantibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp.1547-1553, 1992); using “diabody” technology for making bispecificantibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci.USA, vol. 90, pp. 6444-6448, 1993); and using single-chain Fv (scFv)dimers (see, e.g. Gruber et al., J. Immunol., vol. 152, pp. 5368-5374,1994); and preparing trispecific antibodies as described, e.g., in Tuttet al. J. Immunol., vol. 147, pp. 60-69, 1991.

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or antibody fragment may also include a “Dual Acting Fab”or “DAF” comprising an antigen binding site that binds to Ror2 as wellas another, different antigen (such as Ror1, see, US 2008/0069820, forexample).

The anti-Ror2 antibodies or antibody fragments of the invention may beproduced using recombinant methods and compositions, which are describedin detail in US 2016/0017040.

The physical/chemical properties and/or biological activities of theanti-Ror2 antibodies or antibody fragments of the invention may betested and measured by various assays known in the art. Some of theseassays are described in U.S. Pat. No. 8,853,369.

B. Immunoconjugates

In another aspect, the invention also provides immunoconjugatescomprising an anti-Ror2 antibody or antibody fragment conjugated to oneor more cytotoxic agents, such as chemotherapeutic agents or drugs,growth inhibitory agents, toxins (e.g., protein toxins, enzymaticallyactive toxins of bacterial, fungal, plant, or animal origin, orfragments thereof), or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody or antibody fragment is conjugated to one ormore drugs, including but not limited to a maytansinoid (see U.S. Pat.Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); anauristatin such as monomethylauristatin drug moieties DE and DF (MMAEand MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); adolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos.5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296; Hinman et al., Cancer Res., vol. 53, pp.3336-3342, 1993; and Lode et al., Cancer Res., vol. 58, pp. 2925-2928,1998); an anthracycline such as daunomycin or doxorubicin (see Kratz etal., Current Med. Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al.,Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362, 2006; Torgov etal., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc.Natl. Acad. Sci. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al.,Bioorg. & Med. Chem. Letters, vol. 12, vol. 1529-1532, 2002; King etal., J. Med. Chem., vol. 45, pp. 4336-4343, 2002; and U.S. Pat. No.6,630,579); methotrexate; vindesine; a taxane such as docetaxel,paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; andCC1065.

In another embodiment, an immunoconjugate comprises an antibody orantibody fragment as described herein conjugated to an enzymaticallyactive toxin or fragment thereof, including but not limited todiphtheria A chain, nonbinding active fragments of diphtheria toxin,exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody orantibody fragment as described herein conjugated to a radioactive atomto form a radioconjugate. A variety of radioactive isotopes areavailable for the production of radioconjugates. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Fp²¹² and radioactiveisotopes of Lu. When the radioconjugate is used for detection, it maycomprise a radioactive atom for scintigraphic studies, for example tc99mor I123, or a spin label for nuclear magnetic resonance (NMR) imaging(also known as magnetic resonance imaging, MRI), such as iodine-123again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody/antibody fragment and cytotoxic agent may bemade using a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, vol. 238, pp.1098-, 1987. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res., vol. 52, pp.127-131, 1992; U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates herein expressly contemplate, but are not limitedto conjugates prepared with cross-linker reagents including, but notlimited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB,SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

An exemplary embodiment of an ADC comprises an antibody or antibodyfragment (Ab) which targets a tumor cell, a drug moiety (D), and alinker moiety (L) that attaches Ab to D. In some embodiments, theantibody is attached to the linker moiety (L) through one or more aminoacid residues, such as lysine and/or cysteine.

An exemplary ADC has Formula I as Ab-(L-D)_(p), where p is 1 to about20. In some embodiments, the number of drug moieties that can beconjugated to an antibody is limited by the number of free cysteineresidues. In some embodiments, free cysteine residues are introducedinto the antibody amino acid sequence by the methods described herein.Exemplary ADC of Formula I include, but are not limited to, antibodiesthat have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon et al.,Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments,one or more free cysteine residues are already present in an antibody,without the use of engineering, in which case the existing free cysteineresidues may be used to conjugate the antibody to a drug. In someembodiments, an antibody is exposed to reducing conditions prior toconjugation of the antibody in order to generate one or more freecysteine residues.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more moieties such as drug moieties (D) to anantibody or antibody fragment (Ab) to form an immunoconjugate such as anADC of the Formula I. In some embodiments, ADCs can be prepared using aLinker having reactive functionalities for covalently attaching to thedrug and to the antibody. For example, in some embodiments, a cysteinethiol of an antibody or antibody fragment (Ab) can form a bond with areactive functional group of a linker or a drug-linker intermediate tomake an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, a-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al, Bioconjugate Chemistry, vol. 15,pp. 765-773, 2004.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linkercomponents are known in the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research, vol. 52,pp. 127-131, 1992; U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II as-A_(a)-W_(w)—Y_(y)—, wherein A is a “stretcher unit”, and a is aninteger from 0 to 1; W is an “amino acid unit”, and w is an integer from0 to 12; Y is a “spacer unit”, and y is 0, 1, or 2. An ADC comprisingthe linker of Formula II has the Formula I(A):Ab-(A_(a)-W_(w)—Y_(y)-D)_(p), wherein Ab, D, and p are defined as abovefor Formula I. Exemplary embodiments of such linkers are described inU.S. Pat. No. 7,498,298.

In some embodiments, a linker component comprises a “stretcher unit” (A)that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”(W). In some such embodiments, the amino acid unit allows for cleavageof the linker by a protease, thereby facilitating release of the drugfrom the immunoconjugate upon exposure to intracellular proteases, suchas lysosomal enzymes (Doronina et al., Nat. Biotechnol., vol. 21, pp.778-784, 2003). Exemplary amino acid units include, but are not limitedto, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplarydipeptides include, but are not limited to, valine-citrulline (vc orval-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine(fk or phe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schroder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker component comprises a “spacer unit” (Y)that links the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. Expert Opin. Ther. Patents, vol. 15, pp. 1087-1103,2005). In some embodiments, the spacer unit comprisesp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or-cyano; m is an integer ranging from 0 to 4; X may be one or moreadditional spacer units or may be absent; and p ranges from 1 to about20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to4. Nonlimiting exemplary X spacer units include:

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl. Insome embodiments, R₁ and R₂ are each —CH₃.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al., Bioorg. Med. Chem. Lett., vol. 9, p. 2237-, 1999) and ortho-or para-aminobenzylacetals. In some embodiments, spacers can be usedthat undergo cyclization upon amide bond hydrolysis, such as substitutedand unsubstituted 4-aminobutyric acid amides (Rodrigues et al.,Chemistry Biology, vol. 2, pp. 223-, 1995), appropriately substitutedbicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer.Chem. Soc., vol. 94, p. 5815-, 1972) and 2-aminophenylpropionic acidamides (Amsberry et al, J. Org. Chem., vol. 55, p. 5867, 1990). Linkageof a drug to the a-carbon of a glycine residue is another example of aself-immolative spacer that may be useful in ADCs (Kingsbury et al., J.Med. Chem., vol. 27, p. 144′7, 1984).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al. Bioorganic &Medicinal Chemistry Letters, vol. 12, pp. 2213-2215, 2002; Sun et al.,Bioorganic & Medicinal Chemistry, vol. 11, pp. 1761-1768, 2003).Dendritic linkers can increase the molar ratio of drug to antibody, i.e.loading, which is related to the potency of the ADC. Thus, where anantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula

wherein R₁ and R₂ are independently selected from H and C₁-C₆ alkyl. Insome embodiments, R₁ and R₂ are each —CH₃.

wherein n is 0 to 12. In some embodiments, n is 2 to 10. In someembodiments, n is 4 to 8.

Further nonlimiting exemplary ADCs include the structures:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I.

The compounds of the invention expressly contemplate, but are notlimited to, ADCs prepared with the following linker reagents:bis-maleimido-trioxyethylene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(c-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al., J. Org.Chem., vol. 67, pp. 1866-1872, 2002; Dubowchik, et al., TetrahedronLetters, vol. 38, pp. 5257-60, 1997; Walker, I Org. Chem., vol. 60, pp.5352-5355, 1995; Frisch et al., Bioconjugate Chem., vol. 7, pp. 180-186,1995; U.S. Pat. No. 6,214,345; WO 02/088172; US2003130189; US2003096743;WO 03/026577; WO 03/043583; and WO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyl di ethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Exemplary Drug Moieties 1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are derivatives ofmaytansine, and are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos.4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties areknown in the art and can be isolated from natural sources according toknown methods or produced using genetic engineering techniques (see,e.g., Yu et al., PNAS, vol. 99, pp. 7968-7973, 2002). Maytansinoids mayalso be prepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to,those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat.No. 4,256,746) (prepared, for example, by lithium aluminum hydridereduction of ansamytocin P2); C-20-hydroxy (orC-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared, for example, by demethylation using Streptomyces orActinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared,for example, by acylation using acyl chlorides), and those havingmodifications at other positions of the aromatic ring.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, forexample, by the reaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy/acyloxy(U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion ofmaytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and4,315,929) (for example, isolated from Trewia nudlflora);C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, forexample, by the demethylation of maytansinol by Streptomyces); and4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinoid compounds are useful as the linkageposition. For example, an ester linkage may be formed by reaction with ahydroxyl group using conventional coupling techniques. In someembodiments, the reaction may occur at the C-3 position having ahydroxyl group, the C-14 position modified with hydroxymethyl, the C-15position modified with a hydroxyl group, and the C-20 position having ahydroxyl group. In some embodiments, the linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Maytansinoid drug moieties include those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid drug moiety to a linker of an ADC. Each R mayindependently be H or a C₁-C₆ alkyl. The alkylene chain attaching theamide group to the sulfur atom may be methanyl, ethanyl, or propyl,i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020;Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; Liu et al., Proc.Nall. Acad. Sci. USA, vol. 93, pp. 8618-8623, 1996).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe ADC of the invention, i.e. any combination of R and S configurationsat the chiral carbons (U.S. Pat. Nos. 7,276,497; 6,913,748; 6,441,163;U.S. Pat. No. 633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al(2006) J. Med. Chem. 49:4392-4408. In some embodiments, the maytansinoiddrug moiety has the following stereochemistry:

Exemplary embodiments of maytansinoid drug moieties include, but are notlimited to, DM1; DM3; and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate.

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1 to about 20. In someembodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0425 235 B1. See also Liu et al., Proc. Natl. Acad. Sci. USA, vol. 93,pp. 8618-8623, 1996; and Chari et al., Cancer Research, vol. 52, pp.127-131, 1992.

In some embodiments, antibody-maytansinoid conjugates may be prepared bychemically linking an antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020. Insome embodiments, ADC with an average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody. In some instances, even one molecule oftoxin/antibody is expected to enhance cytotoxicity over the use of nakedantibody.

Exemplary linking groups for making antibody-maytansinoid conjugatesinclude, for example, those described herein and those disclosed in U.S.Pat. No. 5,208,020; EP Patent 0 425 235 B1; Chari et al., CancerResearch, vol. 52, pp. 127-131, 1992; US 2005/0276812 A1; and US2005/016993 A1.

2) Auristatins and Dolastatins

Drug moieties include dolastatins, auristatins, and analogs andderivatives thereof (U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237;6,124,431). Auristatins are derivatives of the marine mollusk compounddolastatin-10. While not intending to be bound by any particular theory,dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al., Antimicrob. Agents and Chemother., vol. 45, pp.3580-3584, 2001) and have anticancer (U.S. Pat. No. 5,663,149) andantifungal activity (Pettit et al., Antimicrob. Agents Chemother., vol.42, pp. 2961-2965, 1998). The dolastatin/auristatin drug moiety may beattached to the antibody through the N (amino) terminus or the C(carboxyl) terminus of the peptidic drug moiety (WO 02/088172; Doroninaet al., Nature Biotechnology, vol. 21, pp. 778-784, 2003; Francisco etal., Blood, vol. 102, pp. 1458-1465, 2003).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties D_(E) and D_(F), disclosed in U.S.Pat. Nos. 7,498,298 and 7,659,241:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

-   -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle),        C₃-C₈heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C_(r) C₈ alkyl-(C₃-C₈ carbocycle),        C₃-C₈heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)—wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C_(r) C₈ alkyl-(C₃-C₈ carbocycle),        C₃-C₈heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle); each R⁸ is        independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈ carbocycle        and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of e is independently H, COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of e is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH;    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₅        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂ and, R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(E) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Nonlimiting exemplary embodiments of ADCs of Formula I comprising MMAFand various linker components further include Ab-MC-PAB-MMAF andAb-PAB-MMAF. Immunoconjugates comprising MMAF attached to an antibody bya linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker (Doronina et al.,Bioconjugate Chem., vol. 17, pp. 114-124, 2006). In some suchembodiments, drug release is believed to be effected by antibodydegradation in the cell.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to a liquidphase synthesis method (see, e.g., E. Schroder and K. Lübke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press).Auristatin/dolastatin drug moieties may, in some embodiments, beprepared according to the methods of: U.S. Pat. Nos. 7,498,298;5,635,483; 5,780,588; Pettit et al., J. Am. Chem. Soc., vol. 111, pp.5463-5465, 1998; Pettit et al., Anti-Cancer Drug Design, vol. 13, pp.243-277, 1998; Pettit et al., Synthesis, vol. 6, pp. 719-725, 1996;Pettit et al., J. Chem. Soc. Perkin Trans. vol. 15, pp. 859-863, 1996;and Doronina, Nat. Biotechnol., vol. 21, pp. 778-784, 2003.

In some embodiments, auristatin/dolastatin drug moieties of formulasD_(E) such as MMAE, and D_(E), such as MMAF, and drug-linkerintermediates and derivatives thereof, such as MC-MMAF, MC-MMAE,MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methodsdescribed in U.S. Pat. No. 7,498,298; Doronina et al., BioconjugateChem., vol. 17, pp. 114-124, 2006; and Doronina et al., Nat. Biotech.,vol. 21, pp. 778-784, 2003 and then conjugated to an antibody ofinterest.

3) Calicheamicin

In some embodiments, the immunoconjugate comprises an antibody orantibody fragment conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics, and analogues thereof, are capableof producing double-stranded DNA breaks at sub-picomolar concentrations(Hinman et al., Cancer Research, vol. 53, pp. 3336-3342, 1993; Lode etal., Cancer Research, vol. 58, pp. 2925-2928, 1998). Calicheamicin hasintracellular sites of action but, in certain instances, does notreadily cross the plasma membrane. Therefore, cellular uptake of theseagents through antibody-mediated internalization may, in someembodiments, greatly enhance their cytotoxic effects. Nonlimitingexemplary methods of preparing antibody-drug conjugates with acalicheamicin drug moiety are described, for example, in U.S. Pat. Nos.5,712,374; 5,714,586; 5,739,116; and 5,767,285.

4) Pyrrolobenzodiazepines

In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). Insome embodiments, PDB dimers recognize and bind to specific DNAsequences. The natural product anthramycin, a PBD, was first reported in1965 (Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5793-5795,1965; Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5791-5793,1965). Since then, a number of PBDs, both naturally-occurring andanalogues, have been reported (Thurston et al., Chem. Rev. vol. 1994,pp. 433-465 1994, including dimers of the tricyclic PBD scaffold (U.S.Pat. Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032;7,528,126; 7,557,099). Without intending to be bound by any particulartheory, it is believed that the dimer structure imparts the appropriatethree-dimensional shape for isohelicity with the minor groove of B-formDNA, leading to a snug fit at the binding site (Kohn, In AntibioticsIII. Springer-Verlag, New York, pp. 3-11 (1975); Hurley andNeedham-VanDevanter, Acc. Chem. Res., vol. 19, pp. 230-237, 1986).Dimeric PBD compounds bearing C2 aryl substituents have been shown to beuseful as cytotoxic agents (Hartley et al Cancer Res., vol. 70, pp.6849-6858, 2010; Antonow, J. Med. Chem. vol. 53, pp. 2927-2941, 2010;Howard et al., Bioorganic and Med. Chem. Letters, vol. 19, pp.6463-6466, 2009).

PBD dimers have been conjugated to antibodies and the resulting ADCshown to have anti-cancer properties. Nonlimiting exemplary linkagesites on the PBD dimer include the five-membered pyrrolo ring, thetether between the PBD units, and the N10-C11 imine group (WO2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US2011/0256157; WO 2011/130598).

Nonlimiting exemplary PBD dimer components of ADCs are of:

-   -   and salts and solvates thereof, wherein:    -   the wavy line indicates the covalent attachment site to the        linker;    -   the dotted lines indicate the optional presence of a double bond        between C1 and C2 or C2 and C3;    -   R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR,        ═CH—R^(D), ═C(R^(D))₂, O—SO₂—R, CO₂R and COR, and optionally        further selected from halo or dihalo, wherein R^(D) is        independently selected from R, CO₂R, COR, CHO, CO₂H, and halo;    -   R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR,        NH₂, NHR, NRR′, NO₂, Me₃Sn and halo;    -   R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂,        NHR, NRR′, NO₂, Me₃Sn and halo;    -   Q is independently selected from 0, S and NH;    -   R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal        cation;    -   R and R′ are each independently selected from optionally        substituted C₁₋₈ alkyl, C₁₋₁₂ alkyl, C₃₋₈ heterocyclyl, C₃₋₂₀        heterocycle, and C₅₋₂₀ aryl groups, and optionally in relation        to the group NRR′, R and R′ together with the nitrogen atom to        which they are attached form an optionally substituted 4-, 5-,        6- or 7-membered heterocyclic ring;    -   R¹², R¹⁶, R¹⁹ and R¹⁷ are as defined for R², R⁶, R⁹ and R⁷        respectively;    -   R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by        one or more heteroatoms, e.g. O, S, N(H), NMe and/or aromatic        rings, e.g. benzene or pyridine, which rings are optionally        substituted; and    -   X and X′ are independently selected from O, S and N(H).

In some embodiments, R and R′ are each independently selected fromoptionally substituted C₁₋₁₂ alkyl, C₃₋₂₀heterocycle, and C₅₋₂₀ arylgroups, and optionally in relation to the group NRR′, R and R′ togetherwith the nitrogen atom to which they are attached form an optionallysubstituted 4-, 5-, 6- or 7-membered heterocyclic ring. In someembodiments, R⁹ and R¹⁹ are H. In some embodiments, R⁶ and R¹⁶ are H.

In some embodiments, R⁷ are R¹⁷ are both OR^(7A), where R^(7A) isoptionally substituted C₁₋₄ alkyl. In some embodiments, R^(7A) is Me. Insome embodiments, R^(7A) is Ch₂Ph, where Ph is a phenyl group. In someembodiments, X is O. In some embodiments, R¹¹ is H. In some embodiments,there is a double bond between C₂ and C3 in each monomer unit.

In some embodiments, R² and R¹² are independently selected from H and R.In some embodiments, R² and R¹²are independently R. In some embodiments,R² and R¹² are independently optionally substituted C₅₋₂₀ aryl orC₅₋₇aryl or C₈₋₁₀ aryl. In some embodiments, R² and R¹² areindependently optionally substituted phenyl, thienyl, napthyl, pyridyl,quinolinyl, or isoquinolinyl. In some embodiments, R² and R¹² areindependently selected from ═O, ═CH₂, =CH—R^(D), and ═C(R^(D))₂. In someembodiments, R² and R¹² each=CH₂. In some embodiments, R² and R¹² areeach H. In some embodiments, R² and R¹² are each ═O. In someembodiments, R² and R¹² are each ═CF₂. In some embodiments, R² and/orR¹² are independently ═C(R^(D))₂. In some embodiments, R² and/or R¹²areindependently ═CH—R^(D).

In some embodiments, when R² and/or R¹² is =CH—R^(D), each group mayindependently have either configuration shown below:

In some embodiments, a ═CH—R^(D) is in configuration (I). In someembodiments, R″ is a C3 alkylene group or a C₅ alkylene group.

The linkers of PBD dimer-val-cit-PAB-Ab and the PBD dimer-Phe-Lys-PAB-Abare protease cleavable, while the linker of PBD dimer-maleimide-acetalis acid-labile.

PBD dimers and ADCs comprising PBD dimers may be prepared according tomethods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598.

5) Anthracyclines

In some embodiments, an ADC may comprise anthracycline. Anthracyclinesare antibiotic compounds that exhibit cytotoxic activity. While notintending to be bound by any particular theory, studies have indicatedthat anthracyclines may operate to kill cells by a number of differentmechanisms, including: 1) intercalation of the drug molecules into theDNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis;2) production by the drug of free radicals which then react withcellular macromolecules to cause damage to the cells, and/or 3)interactions of the drug molecules with the cell membrane (see, e.g., C.Peterson et al., “Transport And Storage Of Anthracycline In ExperimentalSystems And Human Leukemia” in Anthracycline Antibiotics In CancerTherapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102). Becauseof their cytotoxic potential anthracyclines have been used in thetreatment of numerous cancers such as leukemia, breast carcinoma, lungcarcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik,in Anthracycline: Current Status And New Developments, p. 11).

Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin,idarubicin, daunomycin, nemorubicin, and derivatives thereof.Immunoconjugates and prodrugs of daunorubicin and doxorubicin have beenprepared and studied (Kratz et al., Current Med. Chem., vol. 13, pp.477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol. 16,pp. 358-362. 1996; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721,2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol. 97, pp. 829-834,2000; Dubowchik et al., Bioorg. & Med. Chem. Letters, vol. 12, pp.1529-1532, 2002; King et al., J. Med. Chem., vol. 45, pp. 4336-4343,2002; EP 0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugateBR96-doxorubicin reacts specifically with the tumor-associated antigenLewis-Y and has been evaluated in phase I and II studies (Saleh et al.,J. Clin. Oncology, vol. 18, pp. 2282-2292, 2000; Ajani et al., CancerJour., vol. 6, pp. 78-81, 2000; Tolcher et al., J. Clin. Oncology, vol.17, pp. 478-484, 1999).

PNU-159682 is a potent metabolite (or derivative) of nemorubicin(Quintieri et al., Clinical Cancer Research, vol. 11, pp. 1608-1617,2005). Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al. Cancer Treat. Rev. vol.17, pp. 133-138, 1990; Ripamonti et al. Brit. J. Cancer, vol. 65, pp.703-707, 1992), including phase II/III trials for hepatocellularcarcinoma (Sun et al., Proceedings of the American Society for ClinicalOncology, vol. 22, Abs1448, 2003; Quintieri, Proceedings of the AmericanAssociation of Cancer Research, vol. 44:1 st Ed, Abs 4649, 2003;Pacciarini et al., Jour. Clin. Oncology, vol. 24, p. 14116, 2006).

Anthracyclines, including PNU-159682, may be conjugated to antibodiesthrough several linkage sites and a variety of linkers (US 2011/0076287;WO2009/099741; US 2010/0034837; WO 2010/009124), including the linkersdescribed herein.

The linker of PNU-159682 maleimide acetal-Ab is acid-labile, while thelinkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab,and PNU-159682-val-cit-PAB-spacer(R¹R²)-Ab are protease cleavable.

6) Other Drug Moieties

Drug moieties also include geldanamycin (Mandler et al., J. Nat. CancerInst., vol. 92, pp. 1573-1581, 2000; Mandler et al., Bioorganic & Med.Chem. Letters, vol. 10, pp. 1025-1028, 2000; Mandler et al.,Bioconjugate Chem., vol. 13, pp. 786-791, 2002); and enzymaticallyactive toxins and fragments thereof, including, but not limited to,diphtheria A chain, nonbinding active fragments of diphtheria toxin,exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, e.g., WO 93/21232.

Drug moieties also include compounds with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. In some embodiments, when an immunoconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example Tc⁹⁹ or 1¹²³, or a spin label for nuclear magnetic resonance(NMR) imaging (also known as magnetic resonance imaging, MRI), such aszirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.Zirconium-89 may be complexed to various metal chelating agents andconjugated to antibodies, e.g., for PET imaging (WO 2011/056983).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, a peptide may be biosynthesized or chemicallysynthesized using suitable amino acid precursors comprising, forexample, one or more fluorine-19 atoms in place of one or morehydrogens. In some embodiments, labels such as Tc⁹⁹, I¹²³, Re¹⁸⁶, Re¹⁸⁸and In¹¹¹ can be attached via a cysteine residue in the antibody. Insome embodiments, yttrium-90 can be attached via a lysine residue of theantibody. In some embodiments, the IODOGEN method (Fraker et al.,Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978) can be used toincorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy”(Chatal, CRC Press 1989) describes certain other methods.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme. In some such embodiments, aprodrug-activating enzyme converts a prodrug (e.g., a peptidylchemotherapeutic agent, see WO 81/01145) to an active drug, such as ananti-cancer drug. Such immunoconjugates are useful, in some embodiments,in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as Serratia protease, thermolysis, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibodies by recombinant DNA techniques well known in the art. See,e.g., Neuberger et al., Nature, vol. 312, pp. 604-608, 1984.

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody use in thepreparation of ADCs from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADCs in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADCs where p is a certain value from ADCs with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADCs with a distribution of one ormore drug moieties attached to an antibody. The average number of drugsper antibody may be calculated from the mixture by a dual ELISA antibodyassay, which is specific for antibody and specific for the drug.Individual ADCs may be identified in the mixture by mass spectroscopyand separated by HPLC, e.g. hydrophobic interaction chromatography (see,e.g., McDonagh et al., Prot. Engr. Design & Selection, vol. 19, pp.299-307, 2006; Hamblen et al., Clin. Cancer Res., vol. 10, pp.7063-7070, 2004). In certain embodiments, a homogeneous ADC with asingle loading value may be isolated from the conjugation mixture byelectrophoresis or chromatography.

d) Certain Methods of Preparing Immunoconjugates

An immunoconjugate that is an ADC of Formula I may be prepared byseveral routes employing organic chemistry reactions, conditions, andreagents known to those skilled in the art, including: (1) reaction of anucleophilic group of an antibody with a bivalent linker reagent to formAb-L via a covalent bond, followed by reaction with a drug moiety D; and(2) reaction of a nucleophilic group of a drug moiety with a bivalentlinker reagent, to form D-L, via a covalent bond, followed by reactionwith a nucleophilic group of an antibody. Exemplary methods forpreparing an ADC of Formula I via the latter route are described in U.S.Pat. No. 7,498,298.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody or antibodyfragment, such as an aldehyde or ketone carbonyl group, with anucleophilic group on a linker reagent or drug. Useful nucleophilicgroups on a linker reagent include, but are not limited to, hydrazide,oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide. In one embodiment, an antibody is modified to introduceelectrophilic moieties that are capable of reacting with nucleophilicsubstituents on the linker reagent or drug. In another embodiment, thesugars of glycosylated antibodies may be oxidized, e.g. with periodateoxidizing reagents, to form aldehyde or ketone groups which may reactwith the amine group of linker reagents or drug moieties. The resultingimine Schiff base groups may form a stable linkage, or may be reduced,e.g. by borohydride reagents to form stable amine linkages. In oneembodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either galactose oxidase or sodium meta-periodate mayyield carbonyl (aldehyde and ketone) groups in the antibody that canreact with appropriate groups on the drug (Hermanson, BioconjugateTechniques). In another embodiment, antibodies containing N-terminalserine or threonine residues can react with sodium meta-periodate,resulting in production of an aldehyde in place of the first amino acid(Geoghegan & Stroh, Bioconjugate Chem., vol. 3, pp. 138-146, 1992; U.S.Pat. No. 5,362,852). Such an aldehyde can be reacted with a drug moietyor linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADCs are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety, are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody or antibody fragment may beconjugated to a “receptor” (such as streptavidin) for utilization intumor pre-targeting wherein the antibody/antibody fragment-receptorconjugate is administered to the patient, followed by removal of unboundconjugate from the circulation using a clearing agent and thenadministration of a “ligand” (e.g., avidin) which is conjugated to acytotoxic agent (e.g., a drug or radionucleotide).

C. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-Ror2 antibodies or antibodyfragments provided herein may be used for detecting the presence of Ror2in a biological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as breast, pancreas,esophagus, lung and/or brain cells or tissue.

A further aspect of the invention relates to an anti-Ror2 antibody orantibody fragment of the invention for diagnosing and/or monitoring acancer or another disease in which Ror2 expression levels are increasedor decreased from a normal physiological level at at least one locationin the body.

In a preferred embodiment, antibodies or antibody fragments of theinvention may be labelled with a detectable molecule or substance, suchas a fluorescent molecule, a radioactive molecule or any other labelknown in the art as above described. For example, an antibody orantibody fragment of the invention may be labelled with a radioactivemolecule. For example, suitable radioactive molecules include but arenot limited to radioactive atoms used for scintigraphic studies such as¹²³I, ¹²⁴I, ¹¹¹In, ¹⁸⁶Re, and ¹⁸⁸Re. Antibodies or antibody fragments ofthe invention may also be labelled with a spin label for nuclearmagnetic resonance (NMR) imaging, such as iodine-123, iodine-131,indium-Ill, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron. Following administration of the antibody, thedistribution of the radiolabeled antibody within the patient isdetected. Any suitable known method can be used. Some non-limitingexamples include, computed tomography (CT), position emission tomography(PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescenceand sonography.

Antibodies or antibody fragments of the invention may be useful fordiagnosing and staging of cancer and diseases associated with Ror2overexpression. Cancers associated with Ror2 overexpression may includesquamous cell cancer, small-cell lung cancer, non-small cell lungcancer, gastric cancer, pancreatic cancer, glial cell tumors such asglioblastoma and neurofibromatosis, cervical cancer, ovarian cancer,liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer,melanoma, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, sarcomas, hematological cancers(leukemias), astrocytomas, and various types of head and neck cancer orother Ror2 expressing or overexpressing hyperproliferative diseases.

Antibodies or antibody fragments of the invention may be useful fordiagnosing diseases other than cancers for which Ror2 expression isincreased or decreased. Both the (soluble or cellular Ror2 forms can beused for such diagnoses. Typically, such diagnostic methods involve useof a biological sample obtained from the patient. As used herein theterm “biological sample” encompasses a variety of sample types obtainedfrom a subject that can be used in a diagnostic or monitoring assay.Biological samples include but are not limited to blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or a tissue culture or cells derived therefrom, and the progenythereof. For example, biological samples include cells obtained from atissue sample collected from an individual suspected of having a cancerassociated with Ror2 overexpression, and in preferred embodiments fromglioma, gastric, lung, pancreatic, breast, prostate, renal, hepatic andendometrial. Biological samples encompass clinical samples, cells inculture, cell supernatants, cell lysates, serum, plasma, biologicalfluid, and tissue samples.

In a particular embodiment, the invention is a method of diagnosing acancer associated with Ror2 overexpression in a subject by detectingRor2 on cells from the subject using the antibody of the invention. Inparticular, said method may include steps of:

(a) contacting a biological sample of a subject with an antibody orantibody fragment according to the invention under conditions suitablefor the antibody or antibody fragment to form complexes with cells inthe biological sample that express Ror2; and(b) detecting and/or quantifying said complexes, whereby detection ofsaid complexes is indicative of a cancer associated with Ror2overexpression.

In order to monitor the progress of a cancer, the method according tothe invention may be repeated at different times, in order to determineif antibody binding to the samples increases or decreases, wherefrom itcan be determined if the cancer has progressed, regressed or stabilized.

In a particular embodiment, the invention is a method of diagnosing adisease associated with the expression or overexpression of Ror2 or adecrease or increase of the soluble form of Ror2. Examples of suchdiseases may include human immune disorders, thrombotic diseases(thrombosis and atherothrombosis), and cardiovascular diseases

In one embodiment, an anti-Ror2 antibody or antibody fragment for use ina method of diagnosis or detection is provided. In a further aspect, amethod of detecting the presence of Ror2 in a biological sample isprovided. In a further aspect, a method of quantifying the amount ofRor2 in a biological sample is provided. In certain embodiments, themethod comprises contacting the biological sample with an anti-Ror2antibody or antibody fragment as described herein under conditionspermissive for binding of the anti-Ror2 antibody or antibody fragment toRor2, and detecting whether a complex is formed between the anti-Ror2antibody or antibody fragment and Ror2. Such a method may be carried outin vitro or in vivo. In one embodiment, an anti-Ror2 antibody orantibody fragment is used to select subjects eligible for therapy. Insome embodiments, the therapy will include administration of ananti-Ror2 antibody or antibody fragment to the subject.

In certain embodiments, labeled anti-Ror2antibodies or antibodyfragments are provided. Labels include, but are not limited to, labelsor moieties that are detected directly (such as fluorescent,chromophoric, electron-dense, chemiluminescent, and radioactive labels),as well as moieties, such as enzymes or ligands, that are detectedindirectly, e.g., through an enzymatic reaction or molecularinteraction. Exemplary labels include, but are not limited to, theradioisotopes ³²P, ¹⁴C, ¹²⁵I, ¹³H and ¹³¹I fluorophores such as rareearth chelates or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

D. Pharmaceutical Formulations

The anti-Ror2 antibodies or antibody fragments have cell killingactivity. This cell killing activity extends to multiple different typesof cell lines. Further, these antibodies or antibody fragments, onceconjugated to a cytotoxic agent, can reduce tumor size and may exhibitreduced toxicity. See Example 2 of this application. Thus, the anti-Ror2antibodies, fragments or immunoconjugates thereof may be useful fortreating proliferative diseases associated with Ror2 expression. Theantibodies, fragments or immunoconjugates may be used alone or incombination with any suitable agent or other conventional treatments.

The anti-Ror2 antibody or antibody fragment may be used to treatdiseases associated with Ror2 expression, overexpression or activation.There are no particular limitations on the types of cancer or tissuethat can be treated other than the requirement for Ror2 expression.Examples include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastric cancer, pancreatic cancer, glial cell tumorssuch as glioblastoma and neurofibromatosis, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, melanoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, renal cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, sarcomas, hematologicalcancers (leukemias), astrocytomas, and various types of head and neckcancer. More preferable cancers are glioma, gastric, lung, pancreatic,breast, prostate, renal, hepatic and endometrial cancer.

Anti-Ror2 antibodies or antibody fragments are potential activators ofthe innate immune response and thus may be used in the treatment ofhuman immune disorders, such as sepsis. The anti-Ror2 antibody orantibody fragment of the invention may also be used as adjuvants forimmunization such as for vaccines and as anti-infection agents against,for example, bacteria, viruses and parasites.

Anti-Ror2 antibody or antibody fragment may be used to protect against,prevent or treat thrombotic diseases such as venous and arterialthrombosis and atherothrombosis. Anti-Ror2 antibody or antibody fragmentmay also be used to protect against, prevent or treat cardiovasculardiseases as well as to prevent or inhibit the entry of viruses such asLassa and Ebola viruses and to treat viral infections.

In each of the embodiments of the treatment methods described herein,the anti-Ror2 antibody, antibody fragment or anti-Ror2 antibody orantibody fragment immunoconjugate may be delivered in a mannerconsistent with conventional methodologies associated with management ofthe disease or disorder for which treatment is sought. In accordancewith the disclosure herein, an effective amount of the antibody,antibody fragment or immunoconjugate is administered to a subject inneed of such treatment for a time and under conditions sufficient toprevent or treat the disease or disorder. Thus, an aspect of theinvention relates to a method for treating a disease associated with theexpression of Ror2 comprising administering to a subject in need thereofwith a therapeutically effective amount of an antibody, antibodyfragment or immunoconjugate of the invention.

For administration, the anti-Ror2 antibody, antibody fragment orimmunoconjugate may be formulated as a pharmaceutical composition. Thepharmaceutical composition including anti-Ror2 antibody, antibodyfragment or immunoconjugate can be formulated according to known methodsfor preparing pharmaceutical compositions. In such methods, thetherapeutic molecule is typically combined with a mixture, solution orcomposition containing a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier is a material that can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablepharmaceutically acceptable carriers are well-known to those in the art.(See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (MackPublishing Company, 19th ed. 1995)) Formulations may further include oneor more excipients, preservatives, solubilizers, buffering agents,albumin to prevent protein loss on vial surfaces, etc.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc. These considerations can be taken intoaccount by a skilled person to formulate suitable pharmaceuticalcompositions. The pharmaceutical compositions of the invention can beformulated for topical, oral, parenteral, intranasal, intravenous,intramuscular, subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition of, forexample, sterilized water or physiological saline, permit theconstitution of injectable solutions.

In some embodiments, tonicity agents, sometimes known as “stabilizers”are present to adjust or maintain the tonicity of a liquid in acomposition. When used with large, charged biomolecules such as proteinsand antibodies, they are often termed “stabilizers” because they caninteract with the charged groups of the amino acid side chains, therebylessening the potential for inter- and intra-molecular interactions.Tonicity agents can be present in any amount of from 0.1% to 25% byweight, preferably 1 to 5% of the pharmaceutical composition. Preferredtonicity agents include polyhydric sugar alcohols, preferably trihydricor higher sugar alcohols, such as glycerin, erythritol, arabitol,xylitol, sorbitol and mannitol.

Additional excipients include agents which can serve as one or more ofthe following: (1) bulking agents, (2) solubility enhancers, (3)stabilizers and (4) and agents preventing denaturation or adherence tothe container wall. Such excipients may include: polyhydric sugaralcohols (enumerated above); amino acids such as alanine, glycine,glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine,2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugaralcohols such as sucrose, lactose, lactitol, trehalose, stachyose,mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol,galactose, galactitol, glycerol, cyclitols (e.g., inositol),polyethylene glycol; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,α-monothioglycerol and sodium thio sulfate; low molecular weightproteins such as human serum albumin, bovine serum albumin, gelatin orother immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose,glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharidessuch as raffinose; and polysaccharides such as dextrin or dextran.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe employed to help solubilize the therapeutic agent as well as toprotect the therapeutic protein against agitation-induced aggregation,which also permits the formulation to be exposed to shear surface stresswithout causing denaturation of the active therapeutic protein orantibody. Non-ionic surfactants may be present in a concentration rangeof about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml toabout 0.2 mg/ml.

Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80,etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®,polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.),lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acidester, methyl celluose and carboxymethyl cellulose. Anionic detergentsthat can be used include sodium lauryl sulfate, dioctyle sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents includebenzalkonium chloride or benzethonium chloride

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment. To prepare pharmaceutical compositions,an effective amount of the antibody or antibody fragment may bedissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in a water suitably mixed with asurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The anti-Ror2 antibody or antibody fragment can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with one ormore of the other ingredients enumerated above, as may be required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of dimethyl sulfoxide(DMSO) as solvent is envisioned to result in extremely rapidpenetration, delivering high concentrations of the active agents to asmall tumor area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

The antibodies or antibody fragments may be formulated within atherapeutic mixture to deliver about 0.0001 to 10.0 milligrams, or about0.001 to 5 milligrams, or about 0.001 to 1 milligrams, or about 0.001 to0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams perdose. Multiple doses can also be administered at selected timeintervals.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g. tablets or other solids for oraladministration; time release capsules; and any other form currentlyused.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the introduction of antibodies or antibody fragmentsinto host cells. The formation and use of liposomes and/or nanoparticlesare known to those of skill in the art.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) are generally designedusing polymers able to degrade in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beeasily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs)). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations

Pharmaceutical formulations containing an anti-Ror2 antibody or antibodyfragment as described herein are prepared by mixing such antibody orantibody fragment having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG).

Exemplary pharmaceutically acceptable carriers herein further includeinterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated. Preferably,ingredients with complementary activities that do not adversely affecteach other may be combined into a single formulation. For example, itmay be desirable to provide an EGFR antagonist (such as erlotinib), ananti-angiogenic agent (such as a VEGF antagonist which may be ananti-VEGF antibody) or a chemotherapeutic agent (such as a taxoid or aplatinum agent) in addition to the anti-Ror2 antibody, antibody fragmentor immunoconjugate of the present invention. Such active ingredients aresuitably present in combination in amounts that are effective for thepurpose intended.

Active ingredients may be encapsulated in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization.For example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsionsmay be employed. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or antibody fragment, whichmatrices may be in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

E. Therapeutic Methods and Compositions

Any of the anti-Ror2 antibodies or antibody fragments provided hereinmay be used in therapeutic methods. In one aspect, an anti-Ror2 antibodyor antibody fragment for use as a medicament is provided. In furtheraspects, an anti-Ror2 antibody or antibody fragment for use in treatingcancer (e.g., breast cancer, non-small cell lung cancer, pancreaticcancer, brain cancer, cancer of pancreas, brain, kidney, ovary, stomach,leukemia, uterine endometrium, colon, prostate, thyroid, liver,osteosarcoma, and/or melanoma) is provided. In certain embodiments, ananti-Ror2 antibody or antibody fragment for use in a method of treatmentis provided. In certain embodiments, the invention provides an anti-Ror2antibody or antibody fragment for use in a method of treating anindividual having cancer comprising administering to the individual aneffective amount of the anti-Ror2 antibody or antibody fragment. Incertain embodiments, the invention provides an anti-Ror2 antibody orantibody fragment for use in a method of treating an individual havingan immune disorder (e.g., an autoimmune disorder), a cardiovasculardisorder (e.g., atherosclerosis, hypertension, thrombosis), aninfectious disease (e.g., Ebola virus, Marburg virus) or diabetes,comprising administering to the individual an effective amount of theanti-Ror2 antibody or antibody fragment. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow. In further embodiments, the invention provides an anti-Ror2antibody or antibody fragment for use in inhibiting angiogenesis,inhibiting cell proliferation, inhibiting immune function, inhibitinginflammatory cytokine secretion (e.g., from tumor-associatedmacrophages), inhibiting tumor vasculature (e.g., intratumoralvasculature or tumor-associated vasculature), and/or inhibiting tumorstromal function.

In certain embodiments, the invention provides an anti-Ror2 antibody orantibody fragment for use in a method of inhibiting angiogenesis,inhibiting cell proliferation, inhibiting immune function, inhibitinginflammatory cytokine secretion (e.g., from tumor-associatedmacrophages), inhibiting tumor vasculature (e.g., intratumoralvasculature or tumor-associated vasculature), and/or inhibiting tumorstromal function in an individual comprising administering to theindividual an effective of the anti-Ror2 antibody or antibody fragmentto inhibit angiogenesis, inhibit cell proliferation, inhibit immunefunction, inhibit inflammatory cytokine secretion (e.g., fromtumor-associated macrophages), inhibit tumor vasculature development(e.g., intratumoral vasculature or tumor-associated vasculature), and/orinhibit tumor stromal function. An “individual” according to any of theabove embodiments is preferably a human.

In a further aspect, the invention provides for the use of an anti-Ror2antibody or antibody fragment in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment of cancer(in some embodiments, breast cancer, non-small cell lung cancer,pancreatic cancer, brain cancer, cancer of the pancreas, brain, kidney,ovary, stomach, leukemia, uterine endometrium, colon, prostate, thyroid,liver, osteosarcoma, and/or melanoma). In a further embodiment, themedicament is for use in a method of treating cancer comprisingadministering to an individual having cancer an effective amount of themedicament. In a further embodiment, the medicament is for use in amethod of treating an immune disorder (e.g., an autoimmune disorder), acardiovascular disorder (e.g., atherosclerosis, hypertension,thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus) ordiabetes, comprising administering to the individual an effective amountof the anti-Ror2 antibody or antibody fragment. In one such embodiment,the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, e.g., asdescribed below. In a further embodiment, the medicament is forinhibiting angiogenesis, inhibiting cell proliferation, inhibitingimmune function, inhibiting inflammatory cytokine secretion (e.g., fromtumor-associated macrophages), inhibiting tumor vasculature (e.g.,intratumoral vasculature or tumor-associated vasculature), and/orinhibiting tumor stromal function. In a further embodiment, themedicament is for use in a method of inhibiting angiogenesis, inhibitingcell proliferation, inhibiting immune function, inhibiting inflammatorycytokine secretion (e.g., from tumor-associated macrophages), inhibitingtumor vasculature (e.g., intratumoral vasculature or tumor-associatedvasculature), and/or inhibiting tumor stromal function in an individualcomprising administering to the individual an amount effective of themedicament to inhibit angiogenesis, inhibit cell proliferation, promoteimmune function, induce inflammatory cytokine section (e.g., fromtumor-associated macrophages), inhibit tumor vasculature development(e.g., intratumoral vasculature or tumor-associated vasculature), and/orinhibit tumor stromal function. An “individual” according to any of theabove embodiments may be a human.

In a further aspect, the invention provides a method for treating acancer. In one embodiment, the method comprises administering to anindividual having such cancer an effective amount of an anti-Ror2antibody or antibody fragment. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, as described below. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating animmune disorder (e.g., an autoimmune disorder), a cardiovasculardisorder (e.g., atherosclerosis, hypertension, thrombosis), aninfectious disease (e.g., Ebola virus, Marburg virus) or diabetes. Inone such embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for inhibitingangiogenesis, inhibiting cell proliferation, inhibiting immune function,inhibiting inflammatory cytokine secretion (e.g., from tumor-associatedmacrophages), inhibiting tumor vasculature (e.g., intratumoralvasculature or tumor-associated vasculature), and/or inhibiting tumorstromal function in an individual. In one embodiment, the methodcomprises administering to the individual an effective amount of ananti-Ror2 antibody or antibody fragment to inhibit angiogenesis, inhibitcell proliferation, promote immune function, induce inflammatorycytokine section (e.g., from tumor-associated macrophages), inhibittumor vasculature development (e.g., intratumoral vasculature ortumor-associated vasculature), and/or inhibit tumor stromal function. Inone embodiment, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-Ror2 antibodies or antibody fragmentsprovided herein, e.g., for use in any of the above therapeutic methods.In one embodiment, a pharmaceutical formulation comprises any of theanti-Ror2 antibodies or antibody fragments provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical formulation comprises any of the anti-Ror2 antibodies orantibody fragments provided herein and at least one additionaltherapeutic agent, e.g., as described below.

In each and every treatment described above, the antibodies or antibodyfragments of the invention can be used alone, as immunoconjugates or incombination with other agents in a therapy. For instance, an antibody ofthe invention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is an anti-angiogenic agent. In certain embodiments, an additionaltherapeutic agent is a VEGF antagonist (in some embodiments, ananti-VEGF antibody, for example bevacizumab). In certain embodiments, anadditional therapeutic agent is an EGFR antagonist (in some embodiment,erlotinib). In certain embodiments, an additional therapeutic agent is achemotherapeutic agent and/or a cytostatic agent. In certainembodiments, an additional therapeutic agent is a taxoid (e.g.,paclitaxel) and/or a platinum agent (e.g., carboplatinum). In certainembodiments the additional therapeutic agent is an agents that enhancesthe patient's immunity or immune system.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or antibody fragment can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies or antibody fragments canalso be used in combination with radiation therapy.

Antibodies or antibody fragments may be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody or antibody fragment need not be, but is optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question. The effective amount of such other agents depends on theamount of antibody or antibody fragment present in the formulation, thetype of disorder or treatment, and other factors discussed above. Theseare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or antibody fragment (when used alone or in combination withone or more other additional therapeutic agents) will depend on the typeof disease to be treated, the type of antibody or antibody fragment, theseverity and course of the disease, whether the antibody or antibodyfragment is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody or antibody fragment, and the discretion of the attendingphysician. The antibody or antibody fragment is suitably administered tothe patient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 40 mg/kg of antibodyor antibody fragment can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody or antibodyfragment). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an antibody fragment or animmunoconjugate of the invention in place of or in addition to ananti-Ror2 antibody.

Enhancing the host's immune function to combat tumors is the subject ofincreasing interest. Conventional methods include (i) APC enhancement,such as (a) injection into the tumor of DNA encoding foreign MHCalloantigens, or (b) transfecting biopsied tumor cells with genes thatincrease the probability of immune antigen recognition (e.g., immunestimulatory cytokines, GM-CSF, co-stimulatory molecules B7.1, B7.2) ofthe tumor, (iii) adoptive cellular immunotherapy, or treatment withactivated tumor-specific T-cells. Adoptive cellular immunotherapyincludes isolating tumor-infiltrating host T-lymphocytes, expanding thepopulation in vitro, such as through stimulation by IL-2 or tumor orboth. Additionally, isolated T-cells that are dysfunctional may be alsobe activated by in vitro application of the anti-PD-L1 antibodies of theinvention. T-cells that are so-activated may then be readministered tothe host. One or more of these methods may be used in combination withadministration of the antibody, antibody fragment or immunoconjugate ofthe present invention.

Traditional therapies for cancer include the following: (i) radiationtherapy (e.g., radiotherapy, X-ray therapy, irradiation) or the use ofionizing radiation to kill cancer cells and shrink tumors. Radiationtherapy can be administered either externally via external beamradiotherapy (EBRT) or internally via brachytherapy; (ii) chemotherapy,or the application of cytotoxic drug which generally affect rapidlydividing cells; (iii) targeted therapies, or agents which specificallyaffect the deregulated proteins of cancer cells (e.g., tyrosine kinaseinhibitors imatinib, gefitinib; monoclonal antibodies, photodynamictherapy); (iv) immunotherapy, or enhancement of the host's immuneresponse (e.g., vaccine); (v) hormonal therapy, or blockade of hormone(e.g., when tumor is hormone sensitive), (vi) angiogenesis inhibitor, orblockade of blood vessel formation and growth, and (vii) palliativecare, or treatment directed to improving the quality of care to reducepain, nausea, vomiting, diarrhea and hemorrhage. Pain medication such asmorphine and oxycodone, anti-emetics such as ondansetron and aprepitant,can permit more aggressive treatment regimens.

In the treatment of cancer, any of the previously described conventionaltreatments for the treatment of cancer immunity may be conducted, prior,subsequent or simultaneous with the administration of the anti-Ror2antibodies or antibody fragments. Additionally, the anti-Ror2 antibodiesor antibody fragments may be administered prior, subsequent orsimultaneous with conventional cancer treatments, such as theadministration of tumor-binding antibodies (e.g., monoclonal antibodies,toxin-conjugated monoclonal antibodies) and/or the administration ofchemotherapeutic agents.

F. Articles of Manufacture and Kits

In another aspect of the invention, an article of manufacture containingan anti-Ror2 antibody or antibody fragment and other materials usefulfor the treatment, prevention and/or diagnosis of the disordersdescribed above is provided. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, IV solution bags, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is by itself or combined with another compositioneffective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antibody or antibody fragment of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody or antibody fragment; and(b) a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-Ror2 antibody or antibody fragment.

Finally, the invention also provides kits comprising at least oneantibody or antibody fragment of the invention. Kits containingpolypeptide, antibodies or antibody fragments, or antibody drugconjugate of the invention find use in detecting Ror2 expression(increase or decrease), or in therapeutic or diagnostic assays. Kits ofthe invention can contain an antibody coupled to a solid support, e.g.,a tissue culture plate or beads (e.g., sepharose beads). Kits can beprovided which contain antibodies for detection and quantification ofRor2 in vitro, e.g. in an ELISA or a Western blot. Such antibody usefulfor detection may be provided with a label such as a fluorescent orradiolabel.

The kits further contain instructions on the use thereof. In someembodiments, the instructions comprise instructions required by the U.S.Food and Drug Administration for in vitro diagnostic kits. In someembodiments, the kits further comprise instructions for diagnosing thepresence or absence of cerebrospinal fluid in a sample based on thepresence or absence of Ror2 in said sample. In some embodiments, thekits comprise one or more antibodies or antibody fragments. In otherembodiments, the kits further comprise one or more enzymes, enzymeinhibitors or enzyme activators. In still other embodiments, the kitsfurther comprise one or more chromatographic compounds. In yet otherembodiments, the kits further comprise one or more compounds used toprepare the sample for spectroscopic assay. In further embodiments, thekits further comprise comparative reference material to interpret thepresence or absence of Ror2 according to intensity, color spectrum, orother physical attribute of an indicator.

The following examples are illustrative, but not limiting, of the softgelatin capsules of the present disclosure. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in the field, and which are obvious to those skilled in theart, are within the scope of the disclosure.

EXAMPLES Example 1: Conditionally Active Biological (CAB) AntibodiesAgainst Ror2

Antibodies against human Ror2 were produced in this Example. A humanizedantibody against Ror2 was used as the wild-type antibody to generate theCAB antibodies against Ror2. The DNAs encoding the wild-type antibody(heavy chain and light chain variable regions) were evolved to generatemutant antibody heavy chain and light chain variable region libraries.The mutant heavy chain and light chain variable regions in the librarieswere screened for selective binding affinity to human Ror2 at pH 6.0over pH 7.4 by ELISA (FIGS. 2A-2B and 3A-3B). Simultaneously, theexpression level of the mutant antibodies was also optimized for thepurpose of providing higher yields in a subsequent manufacturingprocess. The screening was done in serum using a FLAG tag because therewere human antibodies in the serum which might cause false positives forthe screening. The generated conditionally active antibodies were foundto have a higher binding affinity to Ror2 at pH 6.0 than their bindingaffinity to the Ror2 at pH 7.4 (Tables 1 and 2).

The CAB antibodies did not show aggregation in a buffer as demonstratedin FIG. 4. A CAB antibody was analyzed by size exclusion chromatography.As shown in FIG. 4, only one peak was detected, demonstrating little orno aggregation of the antibody. FIG. 5 shows that some of the selectedmutant antibodies (scFv) demonstrated a higher binding affinity to Ror2at pH 6.0 than the binding affinity to Ror2 at pH 7.4. In addition, FIG.5 also shows that raising the temperature from room temperature to 60°C. did not significantly alter the selectivity of the antibodies.

The conditionally active antibodies all exhibited high expression levelsas shown in Table 4 below, with the column “Clone” indicating theantibodies and the expression level being shown in the second column inmg/ml.

The clones of these antibodies were sent to a service provider with arequested expression level representing an expected expression level(“amount ordered”). However, the actual expression levels of several ofthese antibodies (“amount delivered”) were very high and exceeded theexpected expression levels. At least three clones had expression levelsthat exceeded the expected expression level (BAP048.7.067-HC-FLAG,BAP048.7C02D09-FLAG, and BAP048.7-A03F01-FLAG).

TABLE 4 Antibodies with high expression levels Clone [mg/mL] estimatedyield actual yield BAP048.1-06-07 4.9 100 137 BAP048.7-06-07 3.7 100 94BAP048.7-C02D09 3.8 30 57 BAP048.7-A03F01 4.4 30 65 BAP048.7-A03C05 4.230 36

The conditionally active antibodies were also assayed using surfaceplasmon resonance (SPR) to measure their on and off rates for binding toRor2. The SPR assay can be used to measure on and off rates for proteinbinding. The in vivo on and off rate (in animals and humans) of theconditionally active antibodies is an important feature.

It was observed that the conditionally active antibodies have a goodbinding activity at pH 6.0 and little or no binding activity at pH 7.4(FIGS. 6A-6B). The SPR assay showed that these same conditionally activeantibodies were highly selective at pH 6.0 as compared to pH 7.4 (FIGS.6A-6B).

Example 2: Anti-Ror2 Antibodies Conjugated to a Model Toxin

The anti-Ror2 antibody of the present invention was conjugated to amodel toxin (e.g., paclitaxel) to produce a conditionally activeantibody-drug conjugate (Ror2-CAB-ADC).

Tumors were induced in mice by injection of MDA-MB-436 tumor cells toproduce xenografted mice. The Ror2-CAB-ADC was then injected into thexenografted mice at a dose of 0.3 or 1 mg/kg once a week for 2 weeks.The controls used in this study included PBS buffer as vehicle and thetoxin alone (paclitaxel). The study showed that the Ror2-CAB-ADCprovided a significantly greater reduction in the size of the tumor, incomparison with the controls (FIGS. 7A-7C). The results for the 0.3mg/kg dosage group are presented in FIG. 7B and the results for the 1mg/kg dosage group are presented in FIG. 7C. This study showed that theanti-Ror2 antibody conjugated with toxin is effective in reducing tumorsize.

Example 3. pH Dependent Binding Affinity of Conditionally ActiveAntibody BAP048

The binding affinity of one of the conditionally active antibodiesidentified by the present invention, BAP048 (or BAP048.7 as shown insome of the figures), was tested by pH titration. The wild-type antibodywas used as control. As shown in FIG. 8, the conditionally activeantibody BAP048 is more active at pH lower than pH 6.5, but less activeat pH 7.0. The wild-type antibody does not show pH dependency for itsbinding affinity.

Example 4. Cross-Species Binding Affinity of Conditionally ActiveAntibody BAP048

The conditionally active antibody BAP048 was selected for conditionalbinding affinity of the human Ror2. This conditionally active antibodywas tested for its binding affinity to three targets: human Ror2(hROR2), cynomolgus Ror2 (cynoROR2), and mouse Ror2 (mROR2) using ELISA(FIG. 9). The conditionally active antibody BAP048 shown almostidentical binding affinity to the human and cynomolgus Ror2, butsignificantly lower binding affinity to mouse Ror2. The EC50 of theconditionally active antibody BAP048 for the three targets werecalculated to be 201.4 ng/ml for human Ror2, 327.1 ng/ml for ocynomolgusRor2, and 7653 ng/ml for the mouse Ror2.

Example 5. Cell Killing by Conditionally Active Antibody BAP048-MMAEConjugates

The conditionally active antibody BAP048 was conjugated to chemotherapydrug Monomethyl auristatin E (MMAE) to produce an antibody drugconjugate (ADC). The ADC was tested on HEK293 cells that express humanRor2 on the cell surface. Negative control used for this test was anaffinity matching antibody but not specific for human Ror2. The negativecontrol was also conjugated to MMAE.

The BAP048 ADC was tested under two pH: 6.0 and 7. The percentage ofcells remained alive after the treatment, relative to the cells treatedby the negative control was presented in FIG. 10. It was observed thatthe BAP048 ADC displayed cell killing activity at pH 6.0 at aconcentration below 100 ng/ml, while at pH 7.4, the BAP048 ADC showedcell killing at about 1000 ng/ml, a difference of effectiveconcentration about 10 fold.

The cell killing activity of the BAP048 ADC was also tested on LCLC103Hcells and HT1080 cells. For LCLC103H cells, which are a human lungcancer line, the cell killing activity of the BAP048 ADC was tested atpH 6.0, 6.2, and 6.4 (FIGS. 11A-11C). The difference of the cell killingactivity of the BAP048 ADC was not significantly different among thethree tested pH values, with the IC50 at 1.819 ng/ml (pH 6.0), 1.232ng/ml (pH 6.2), and 3.318 ng/ml (pH 6.5).

For HT1080 cells, which are a fibrosarcoma cell line, the cell killingactivity of the BAP048 ADC was also tested at pH 6.0, 6.2, and 6.4(FIGS. 12A-12C). The difference of the cell killing activity of theBAP048 ADC was not significantly different among the three tested pHvalues, with the IC50 at 2.897 ng/ml (pH 6.0), 3.138 ng/ml (pH 6.2), and2.601 ng/ml (pH 6.5).

Example 6. Treatment of Tumor in Mice by Conditionally Active AntibodyBAP048-MMAE Conjugates

The lung cancer cells LCLC103H were injected into mice to generate mousezenografts (tumors in mice). The conditionally active antibody BAP048was conjugated to MMAE through a linker thiobridge to generate an ADCfor injection to the mice at two concentrations 1 mg/kg and 6 mg/kg. Thenegative control was the buffer (vehicle) without the ADC. One dosegroup was injected at 1 mg/kg dose weekly for three doses, and the otherdose group was injected at 6 mg/kg dose every 4 days for 4 doses.

The volumes of the tumors were measured during the study. It wasobserved that at 6 mg/kg dose group, the tumor size shrank significantlyby day 30 by the BAP048-MMAE ADC. At the 1 mg/kg dose group, the tumorgrowth was slowed by the BAP048-MMAE ADC, in comparison with the vehiclecontrol (FIG. 13).

Example 7. Effect of Linker in Conditionally Active Antibody BAP048-MMAEConjugates

The effect of the linkers of the conditionally active antibody BAP048MMAE conjugation was tested using two different linkers: mc-vc-PAB andmc-PEG8-vc (FIG. 14). The same negative control (vehicle) was used.These two BAP048-MMAE conjugates were injected into the mouse xenograftsat a single dose of 1 mg/ml. The size of the tumors was measured duringthe study. It was observed that though both BAP048-MMAE conjugatessignificantly slowed the growth of the tumors in comparison with thenegative control, the difference between the two BAP048-MMAE conjugateswas not significant (FIG. 14). Thus, it appears the affect of thelinkers on the BAP048-MMAE conjugates is not significant.

Example 8. Treatment of Tumor in Mice by Conditionally Active AntibodyBAP048

This example is similar to Example 6, except the cells used to inducethe tumors in the mice are different. The two cell lines used in thisexample are HT1080 cells (a fibrosarcoma cell line) and MDA-MB-436 cells(a breast cancer cell line).

For the mouse xenograft induced by HT1080, the conditionally activeantibody BAP048 was conjugated to MMAE with the linker of mc-vc-PAB.Three dose groups were used: 6 mg/kg every 4 days for 4 doses, 10 mg/kgevery week for 3 doses, and 10 mg/kg every 4 days for 4 doses (FIG.15A). Tow controls were used: one is the vehicle without antibody orMMAE, the other is the BAP048 antibody without conjugated MMAE.

It was observed that the tumor grew steadily with the two controlgroups. The growth of the tumor volume was slowed significantly by theconditionally active antibody BAP048-MMAE conjugates. The inhibition ofthe tumor growth was dose dependent, with the highest dose group, 10mg/kg every 4 days for 4 doses, showed the largest reduction in tumorgrowth (FIG. 15A).

For the mouse xenograft induced by MDA-MB-436, the conditionally activeantibody BAP048 was also conjugated to MMAE with the linker ofmc-vc-PAB. There was only one dose group: 6 mg/kg every 4 days for 4doses, with the vehicle (no antibody or MMAE) as the control (FIG. 15B).It was observed that the tumor grew steadily with the control group. Thegrowth of the tumor volume was reduced significantly by theconditionally active antibody BAP048-MMAE conjugates (FIG. 15B).

It is to be understood, however, that even though numerouscharacteristics and advantages of the present invention have been setforth in the foregoing description, together with details of thestructure and function of the invention, the disclosure is illustrativeonly, and changes may be made in detail, especially in matters of shape,size and arrangement of parts within the principles of the invention tothe full extent indicated by the broad general meanings of the terms inwhich the appended claims are expressed.

All documents mentioned herein are hereby incorporated by reference intheir entirety or alternatively to provide the disclosure for which theywere specifically relied upon. The applicant(s) do not intend todedicate any disclosed embodiments to the public, and to the extent anydisclosed modifications or alterations may not literally fall within thescope of the claims, they are considered to be part hereof under thedoctrine of equivalents.

1. An isolated polypeptide that specifically binds to Ror2 protein, saidpolypeptide comprising a heavy chain variable region including threecomplementarity determining regions, said regions having H1, H2, and H3sequences, wherein: (a) the H1 sequence is GYTX₁TEX₂X₃X₄H (SEQ ID NO:1)or GYSITTGX₂₉YWN (SEQ ID NO:4); (b) the H2 sequence isX₅X₆X₇X₈NNGGTGYNQKFKG (SEQ ID NO:2) or YITYDGSX₃₀NYNPSLKN (SEQ ID NO:5);and (c) the H3 sequence is X₉X₁₀X₁₁SX₁₂YX₁₃YX₁₄X₁₅SYFX₁₆X₁₇X₁₈ (SEQ IDNO:3) or CSX₃₁X₃₂X₃₃X₃₄VX₃₅X₃₆X₃₇LDX₃₈ (SEQ ID NO:6); wherein X₁ is F orE, X₂ is Y or D, X₃ is T or C, X₄ is M or D or E or Y, X₅ is G or S, X₆is I or E, X₇ is N or C or L or V, X₈ is T or D or E, X₉ is A or M or T,X₁₀ is R or H, X₁₁ is G or E, X₁₂ is L or F, X₁₃ is S or G, X₁₄ is G orD, X₁₅ is N or E, X₁₆ is D or L, X₁₇ is Y or C or T, X₁₈ is W or L, X₂₉is Y or E or R or T, X₃₀ is K or N, X₃₁ is R or G or H or W or Y, X₃₂ isF or C or N or Q, X₃₃ is E or S, X₃₄ is G or E or F or H or M or Q or S,X₃₅ is W or A or I or P or Q or T or V, X₃₆ is Y or G or N or Q, X₃₇ isG or S or T, and X₃₈ is Y or I.
 2. The polypeptide of claim 1, whereinthe heavy chain variable region has an amino acid sequence selected fromsequences of SEQ ID NOS: 18-26.
 3. The polypeptide of claim 1 incombination with an isolated light chain variable region including threecomplementarity determining regions L1, L2, and L3 sequences, wherein:(a) the L1 sequence is SATSSX₁₉X₂₀X₂₁MX₂₂ (SEQ ID NO:7) orRASESVDRYGNSX₃₉IH (SEQ ID NO:10); (b) L2 sequence is X₂₃TSNLAS (SEQ IDNO:8) or X₄₀TYX₄₁LES (SEQ ID NO:11); and (c) L3 sequence isQX₂₄X₂₅SX₂₆YPFX₂₇X₂₈ (SEQ ID NO:9) or QQX₄₂NX₄₃DPX₄₄TX₄₅ (SEQ ID NO:12);wherein X₁₉ is V or E, X₂₀ is S or D, X₂₁ is Y or C or D, X₂₂ is H or Gor L, X₂₃ is G or C or H or P, X₂₄ is Q or E, X₂₅ is R or H, X₂₆ is S orD or G or I or Q or V, X₂₇ is T or D, X₂₈ is F or D or E, X₃₉ is F or Sor T, X₄₀ is R or C or D or E or W, X₄₁ is N or D, X₄₂ is T or I or P,X₄₃ is E or V, X₄₄ is W or T, and X₄₅ is F or T.
 4. The polypeptide ofclaim 3, wherein the light chain variable region has an amino acidsequence selected from SEQ ID NOS: 13-17 and
 27. 5. The polypeptide ofclaim 1, wherein the X₂₉ is Y.
 6. The polypeptide of claim 1, whereinthe X₂₉ is E.
 7. An isolated polypeptide that specifically binds to Ror2protein, said polypeptide comprising an light chain variable regionincluding three complementarity determining regions having L1, L2, andL3 sequences, wherein: (a) the L1 sequence is SATSSX₁₉X₂₀X₂₁MX₂₂ (SEQ IDNO:7) or RASESVDRYGNSX₃₉IH (SEQ ID NO:10); (b) the L2 sequence isX₂₃TSNLAS (SEQ ID NO:8) or X₄₀TYX₄₁LES (SEQ ID NO:11); and (c) the L3sequence is QX₂₄X₂₅SX₂₆YPFX₂₇X₂₈ (SEQ ID NO:9) or QQX₄₂NX₄₃DPX₄₄TX₄₅(SEQ ID NO:12); wherein X₁₉ is V or E, X₂₀ is S or D, X₂₁ is Y or C orD, X₂₂ is H or G or L, X₂₃ is G or C or H or P, X₂₄ is Q or E, X₂₅ is Ror H, X₂₆ is S or D or G or I or Q or V, X₂₇ is T or D, X₂₈ is F or D orE, X₃₉ is F or S or T, X₄₀ is R or C or D or E or W, X₄₁ is N or D, X₄₂is T or I or P, X₄₃ is E or V, X₄₄ is W or T, and X₄₅ is F or T.
 8. Thepolypeptide of claim 7, wherein the light chain variable region isencoded by a DNA sequence selected from SEQ ID NOS: 13-17 and
 27. 9. Thepolypeptide of claim 7, wherein the light chain variable regionincluding three complementarity determining regions L1, L2, and L3having sequences of SEQ ID NOS: 10-12 respectively.
 10. An anti-Ror2antibody or antibody fragment comprising the isolated heavy chainvariable region polypeptide of claim
 1. 11. The antibody or antibodyfragment of claim 10, further comprising an isolated light chainvariable region comprising three complementarity determining regions L1,L2, and L3 sequences, wherein: (a) the L1 sequence is SATSSX₁₉X₂₀X₂₁MX₂₂(SEQ ID NO:7) or RASESVDRYGNSX₃₉IH (SEQ ID NO:10); (b) the L2 sequenceis X₂₃TSNLAS (SEQ ID NO:8) or X₄₀TYX₄₁LES (SEQ ID NO:11); and (c) the L3sequence is QX₂₄X₂₅SX₂₆YPFX₂₇X₂₈ (SEQ ID NO:9) or QQX₄₂NX₄₃DPX₄₄TX₄₅(SEQ ID NO:12); wherein X₁₉ is V or E, X₂₀ is S or D, X₂₁ is Y or C orD, X₂₂ is H or G or L, X₂₃ is G or C or H or P, X₂₄ is Q or E, X₂₅ is Ror H, X₂₆ is S or D or G or I or Q or V, X₂₇ is T or D, X₂₈ is F or D orE, X₃₉ is F or S or T, X₄₀ is R or C or D or E or W, X₄₁ is N or D, X₄₂is T or I or P, X₄₃ is E or V, X₄₄ is W or T, and X₄₅ is F or T.
 12. Theantibody or antibody fragment of claim 10, wherein the antibody orantibody fragment has a higher binding affinity to Ror2 protein at avalue of a condition in a tumor microenvironment in comparison with adifferent value of the same condition that occurs in a non-tumormicroenvironment.
 13. The antibody or antibody fragment of claim 12,wherein the condition is pH.
 14. The antibody or antibody fragment ofclaim 13, wherein the pH in the tumor microenvironment is in a range offrom 5.8 to 6.8 and the pH in the non-tumor microenvironment is in arange of 7.0 to 7.6.
 15. The antibody or antibody fragment of claim 10,wherein the antibody or antibody fragment has a ratio of bindingaffinity to the Ror2 protein at a value of a condition in a tumormicroenvironment to a binding affinity to the Ror2 protein at adifferent value of the same condition in a non-tumor microenvironment ofat least about 1.5:1, at least about 2:1, at least about 3:1, at leastabout 4:1, at least about 5:1, at least about 6:1, at least about 7:1,at least about 8:1, at least about 9:1, at least about 10:1, at leastabout 20:1, at least about 30:1, at least about 50:1, at least about70:1, or at least about 100:1.
 16. (canceled)
 17. An immunoconjugatecomprising the antibody or antibody fragment of claim
 10. 18. Theimmunoconjugate of claim 17, wherein the immunoconjugate comprises atleast one agent selected from a chemotherapeutic agent, a radioactiveatom, a cytostatic agent and a cytotoxic agent.
 19. (canceled) 20.(canceled)
 21. The immunoconjugate of claim 17, wherein the at least oneagent is selected from maytansinoids, auristatins, dolastatins,calicheamicin, pyrrolobenzodiazepines, and anthracyclines.
 22. Apharmaceutical composition comprising: the antibody or antibody fragmentof claim 10; and a pharmaceutically acceptable carrier.
 23. (canceled)24. A method of treating cancer comprising a step of administering thepharmaceutical composition of claim 22 to a patient with cancer.
 25. Akit for diagnosis or treatment, said kit comprising the antibody orantibody fragment of claim 10 and instructions for using the antibody orantibody fragment for diagnosis or treatment.